combination of insecticides

Abstract

Novel combinations of cysteine-rich insecticidal proteins (CRIPs) and insecticides (IAs), such as chemicals, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxic substances, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes or eukaryotes (and substances produced from such prokaryotes or eukaryotes), are described and claimed for the control and / or eradication of pests. [Solution] Novel insecticide combinations, compositions, and methods for using them are provided. The present invention relates to a combination of a cysteine-rich insecticide peptide (CRIP) and an insecticide (IA). The present invention also describes the use of the combination and composition for controlling insects. This specification describes the gene encoding CRIP, compositions and combinations containing CRIP and IA, and methods useful for controlling pests using them.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 63 / 019,219, filed on 1 May 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence List This application was prepared on 28 April 2021 at 10:07 PM, and the sequence listing titled "225312-491452_ST25.txt" (1.41 MB), filed electronically with this specification, is incorporated by reference in its entirety.

[0003] Novel combinations of cysteine-rich insecticidal proteins (CRIPs) and insecticides (IAs), such as chemicals, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxic substances, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes or eukaryotes (and substances produced from such prokaryotes or eukaryotes), are described and claimed for the control and / or eradication of pests. [Background technology]

[0004] Numerous insects are disease vectors. Mosquitoes of the genus Anopheles are the primary vectors of Zika virus, Chikungunya virus, and malaria, a disease caused by protozoa of the genus Trypanosoma. Aedes aegypti are the primary vectors of the viruses that cause yellow fever and dengue fever. Other viruses that cause various types of encephalitis are also carried by Aedes spp. mosquitoes. Wuchereria bancrofti and Brugia malayi, parasitic roundworms that cause filariasis, are usually spread by mosquitoes of the genera Culex, Mansonia, and Anopheles.

[0005] Horseflies and deerflies can transmit bacterial pathogens such as tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as the parasitic roundworm (Loa loa) that causes loa disease in tropical Africa.

[0006] Eye gnats of the genus Hippelates can carry spirochete pathogens that cause strawberry cysts (Treponema pertenue) and may also spread conjunctivitis (pink eye). Tsetse flies of the genus Glossina transmit protozoan pathogens (Trypanosoma gambiense and T. rhodesiense) that cause African sleeping sickness. Sandflies of the genus Phlebotomus are vectors of the bacterium (Bartonella bacilliformis) that causes Carillon's disease (Oroyo fever) in South America. In parts of Asia and North Africa, they transmit viral pathogens that cause sandfly fever (Papatachi fever), as well as protozoan pathogens (Leishmania spp.) that cause leishmaniasis.

[0007] Therefore, effective pesticide treatment is necessary to preserve the crops on which we depend for food and to protect human and animal health.

[0008] This specification describes combinations of insecticides (IAs) and cysteine-rich insecticidal peptides (CRIPs). An IA is one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxic substances, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes and / or products thereof (e.g., bacterial toxins), or eukaryotes and / or products thereof (e.g., mycotoxins). Combinations of IAs can provide an insecticidal effect greater than the additive effect of any individual IAs used for isolation.

[0009] CRIPs are, in some embodiments, peptides, polypeptides, and / or proteins having cysteine ​​residues capable of forming disulfide bonds, which generate scaffolding motifs observed in a wide variety of unrelated protein families. An example of peptides included in the CRIP family is inhibitor-cystine knot (ICK) peptides. ICK peptides include many molecules with insecticidal activity. Such ICK peptides are often toxic to naturally occurring biological target species (usually certain insects or spiders). Often, ICK peptides may have arthropod origins, such as scorpion or spider venom.

[0010] This specification describes novel insecticide combinations of IA and CRIP. For example, the present invention describes, in particular, insecticidal combinations comprising (1) one or more CRIP or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA), and methods of using them to preserve crops on which we depend for food and to protect human and animal health. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] U.S. Provisional Application No. 63 / 019,219 [Overview of the project] [Means for solving the problem]

[0012] This invention describes a method for combining CRIP and IA to provide a greater insecticidal effect than the additive insecticidal effect of any IA or CRIP used alone. This disclosure describes a method for preparing and using combinations of CRIP and IA to kill and control insects, even at low doses, and even in insecticide-resistant insects. Without being bound by theory, our understanding of CRIP and IA will teach those skilled in the art and enable the creation of novel methods, compositions, compounds (proteins and peptides), and procedures for protecting plants and controlling insects.

[0013] This disclosure describes combinations comprising a cysteine-rich insecticide peptide (CRIP) and an insecticide (IA).

[0014] In addition, this disclosure describes combinations comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), where IA is a bacterial toxin, fungal toxin, lectin, Azadirachta indica compound, boron compound, virus, or a combination thereof, and CRIP is U1-agatoxin-Ta1b peptide, U1-agatoxin-Ta1b variant polypeptide (TVP), anemone toxin, Av3 variant polypeptide (AVP), Phoneutria toxin, or atracotoxin (ACTX).

[0015] Furthermore, this disclosure describes compositions comprising a combination of a cysteine-rich insecticide peptide (CRIP) and an insecticide (IA), further comprising excipients.

[0016] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 and a U1-agatoxin-Ta1b peptide having an amino acid sequence as shown in Sequence ID No. 1.

[0017] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 and a U1-agatoxin-Ta1b variant polypeptide (TVP) having an amino acid sequence as shown in SEQ ID NO: 2.

[0018] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 and an Av3 variant polypeptide (AVP) having an amino acid sequence as shown in SEQ ID NO: 67.

[0019] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 and a Γ-CNTX-Pn1a toxin having an amino acid sequence as shown in SEQ ID NO: 65.

[0020] Furthermore, this disclosure describes a combination comprising Beauveria bassiana strain ANT-03 spores and the U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO: 61.

[0021] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. tenebrionis strain NB-176, and the U+2-ACTX-Hv1a toxin having an amino acid sequence as shown in SEQ ID NO: 61.

[0022] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 and the U+2-ACTX-Hv1a toxin having an amino acid sequence as shown in SEQ ID NO: 61.

[0023] Furthermore, this disclosure describes combinations comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. israelensis strain BMP144, and the U+2-ACTX-Hv1a toxin having an amino acid sequence as shown in SEQ ID NO: 61.

[0024] Furthermore, this disclosure describes a combination comprising Photorhabdus luminescens toxin and ACTX, wherein the Photorhabdus luminescens toxin is a Photorhabdus luminescens toxin complex (Tca) comprising TcaA (SEQ ID NO: 616), TcaB (SEQ ID NO: 617), TcaC (SEQ ID NO: 618), and TcaZ (SEQ ID NO: 619), and the ACTX peptide is U+2-ACTX-Hv1a toxin (SEQ ID NO: 61).

[0025] Furthermore, this disclosure describes a combination comprising Galanthus nivalis agglutinin (GNA) and ACTX, wherein GNA has the amino acid sequence shown in SEQ ID NO: 35, and the ACTX peptide is a U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO: 61.

[0026] Furthermore, this disclosure describes combinations comprising azadirachtin and ACTX, wherein azadirachtin has the chemical formula: C 35 H 44 O 16 ACTX is a U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO: 61.

[0027] Furthermore, this disclosure describes a combination comprising a boric acid compound and ACTX, wherein the boric acid compound has the chemical formula H3BO3 and the ACTX peptide is a U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO: 61.

[0028] Furthermore, this disclosure describes a combination comprising Cydia pomonella granulovirus (CpGV) and ACTX, where CpGV is the Cydia pomonella granulovirus isolated from strain V22, and the ACTX peptide is the U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO: 61.

[0029] Furthermore, this disclosure describes a method for controlling insects using a combination comprising a cysteine-rich insecticide peptide (CRIP) and an insecticide (IA), the method comprising providing a combination of at least one CRIP and at least one IA, and applying the combination comprising the cysteine-rich insecticide peptide (CRIP) and the insecticide (IA) to a site on an insect.

[0030] Furthermore, this disclosure describes a method for controlling toxin-resistant Bacillus thuringiensis insects using a combination of a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), comprising providing a combination of at least one CRIP and at least one IA, and then applying the combination to a site on the insect.

[0031] Furthermore, this disclosure describes methods for eradicating, controlling, or suppressing pests, including applying a pesticide-effective amount of a combination of a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA) to the site of the pest or to plants or animals susceptible to pest attack. [Brief explanation of the drawing]

[0032] [Figure 1]The graph shows the 24-hour mortality rate of Aedes aegypti (mosquito) larvae after a diet incorporation assay using (1) U+2-ACTX-Hv1a with Bti, (2) Bti toxin alone, (3) U+2-ACTX-Hv1a alone, and (4) a control (water). [Figure 2] This graph shows the 3-day mortality rate of Lepidopteran beet armyworms (Spodoptera exigua) after a foliar spray assay using Bacillus thuringiensis var. kurstaki toxin (Btk) combined with Γ-CNTX-Pn1a. The treatments were (1) Γ-CNTX-Pn1a alone, (2) Btk toxin alone, (3) a combination of Γ-CNTX-Pn1a and Btk toxin, or (4) a control (0.125% Vintre, surfactant). [Figure 3] This graph shows the 3-day mortality rate of Lepidopteran beet army worms (Spodoptera exigua) after a foliar spray assay combining Btk and Av3-variant polypeptide (AVP). Here, (1) AVP alone, (2) Btk toxin alone, (3) a combination of both AVP and Btk toxin, or (4) a control (0.125% Vintre, surfactant) were tested. The AVP tested here was AVPb. [Figure 4] Chromatograms evaluating WT-Ta1b degradation in Helicoverpa zea intestinal extract (HGE), a simulated Lepidopteran intestinal environment, at 0, 20, 40, 60, 180, and 1260 minutes. Boxes indicate the primary and secondary peaks, and therefore the degradation of WT-Ta1b. Nested insets show enlarged / reduced views of the chromatograms. Boxes highlight peaks indicating proteolytic events, evidenced by the presence of two shoulders; the smaller "shoulder" to the right of the primary peak demonstrates a partial proteolytic event. [Figure 5]Chromatograms evaluating the degradation of TVP-R9Q in Helicoverpa zea intestinal extract (HGE), a simulated Lepidopteran intestinal environment, at 0, 20, 40, 60, 180, and 1260 minutes are shown. Boxes indicate the presence of a single peak, and therefore, the stability of TVP-R9Q. Nested insets show enlarged / reduced views of the chromatograms. Here, the presence of a single main peak (indicated by a box) indicates the stability of the TVP-R9Q peptide. [Figure 6] The graph shows the results of a leaf fall assay against the Lepidopteran species Helicoverpa zea (corn earworm) when tested with WT-Ta1b, Btk toxin, and combinations thereof. The treatments were as follows: (1) WT-Ta1b alone, (2) Btk toxin alone, (3) a combination of both WT-Ta1b and Btk toxin, or (4) control (0.125% Vintre, surfactant). Here, Btk toxin is indicated as "Btk". [Figure 7] The graph shows the results of a leaf fall assay against the Lepidopteran species Helicoverpa zea (corn ear worm) when tested with TVP-R9Q, Btk toxin, and combinations thereof. The treatments were as follows: (1) TVP-R9Q alone, (2) Btk toxin alone, (3) a combination of both TVP-R9Q and Btk toxin, or (4) control (0.125% Vintre, surfactant). Here, Btk toxin is indicated as "Btk". [Figure 8] The graph shows the results of a mortality assay for the Lepidopteran species Helicoverpa zea (corn ear worm) when tested with WT-Ta1b, Btk toxin, and combinations thereof. The treatments were as follows: (1) WT-Ta1b alone, (2) Btk toxin alone, (3) a combination of both WT-Ta1b and Btk toxin, or (4) control (0.125% Vintre, surfactant). Here, Btk toxin is indicated as "Btk". [Figure 9]The graph shows the results of a mortality assay in Lepidopteran species Helicoverpa zea (corn ear worm) when tested with TVP-R9Q, Btk toxin, and combinations thereof. The treatments were as follows: (1) TVP-R9Q alone, (2) Btk toxin alone, (3) combination of both TVP-R9Q and Btk toxin, or (4) control (0.125% Vintre, surfactant). Here, Btk toxin is indicated as "Btk". [Figure 10] The graph shows the 4-day mortality rate of Coleoptera species darkling beetles (Alphitobius diaperinus) after feed contamination assays using (1) U+2-ACTX-Hv1a alone, (2) Btt toxin alone, (3) a combination of both U+2-ACTX-Hv1a and Btt toxin, or (4) an untreated control (water). [Figure 11] The graph shows the 4-day mortality rate of Colorado potato beetles (Leptinotarsa ​​decemlineata) when (1) U+2-ACTX-Hv1a alone, (2) Btt toxin alone, (3) a combination of both U+2-ACTX-Hv1a and Btt toxin, or (4) an untreated control (water) was sprayed. [Figure 12] The graph below shows the mortality rate on day 4 in corn ear worm larvae treated with the following: (a) water, (b) Photorhabdus luminescens toxin complex extract alone (4.75% v / v), (c) 10 mg / mL U+2-ACTX-Hv1a (1% w / v), and (d) Photorhabdus luminescens toxin complex extract (4.75% w / v) and 10 mg / mL U+2-ACTX-Hv1a (1% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water. [Figure 13]The graphs show the mortality rates of corn ear worm hatched larvae (neonates) 3 days after treatment with (a) 0 mg / mL GNA (0% w / v) and 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control), (b) 2.5 mg / mL GNA (0.25% w / v) and 0 mg / mL U+2-ACTX-Hv1a (0% w / v), (c) 0 mg / mL GNA (0% w / v) and 5 mg / mL U+2-ACTX-Hv1a (0.5% w / v), and (d) 2.5 mg / mL GNA (0.25% w / v) and 5 mg / mL U+2-ACTX-Hv1a (0.5% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water. Proportional mortality refers to the proportion of individual insects that died during the experiment (i.e., the number of dead individuals relative to the total number of individuals). [Figure 14] (a) Chitinase 0 μL / L (0% w / v), U+2-ACTX-Hv1a 0 mg / mL (0% w / v), sucrose (10% w / v), (b) Chitinase 100 μL / L (0.01% w / v), U+2-ACTX-Hv1a 0 mg / mL (0% w / v), sucrose (10% w / v), (c) Chitinase 0 μL / L (0% w / v), U+2-ACTX-Hv1a 5 mg / mL (0.5% w / v), sucrose (10% w / v), and (d) Chitinase 100 μL / L (0.01% w / v), U+2-ACTX-Hv1a 5 mg / mL (0.5% w / v), sucrose (10% The graph shows the mortality rate of fall armyworm (Spodoptera frugiperda) larvae three days after treatment with w / v. Here, % w / v is the percentage of w / v of the total volume of the composition, with the remainder being water. [Figure 15] The chemical structure of azadirachtin, an insect growth regulator, is shown. [Figure 16]The graph shows the mortality rates of corn ear worms (Helicoverpa zea) 3 days after treatment with (a) 0 μL / L azadirachtin (0% v / v) and 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control), (b) 80 μL / L azadirachtin (0.008% v / v) and 0 mg / mL U+2-ACTX-Hv1a (0% w / v), (c) 0 μL / L azadirachtin (0% v / v) and 10 mg / mL U+2-ACTX-Hv1a (1% w / v), and (d) 80 μL / L azadirachtin (0.008% v / v) and 10 mg / mL U+2-ACTX-Hv1a (1% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water. [Figure 17] The following graphs show the mortality rates of hatched larvae of Lesser mealworm (Alphitobius diaperinus) three days after treatment: (a) 0 mg / mL U+2-ACTX-Hv1a (0% w / v), 0 mg / mL boric acid (0% w / v) (control), (b) 0 mg / mL U+2-ACTX-Hv1a (0% w / v), 2.5 mg / mL boric acid (0.25% w / v), (c) 1 mg / mL U+2-ACTX-Hv1a (0.1% w / v), 0 mg / mL boric acid (0% w / v), and (d) 1 mg / mL U+2-ACTX-Hv1a (0.1% w / v), 2.5 mg / mL boric acid (0.25% w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water. [Figure 18]The following graphs show the mortality rates in hatched larvae of Codling Moths (Cydia pomonella) 7 days after treatment: (a) 0 mg / mL Beauveria bassiana toxin (0% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control), (b) 1.2 mg / mL Beauveria bassiana toxin (0.12% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v), (c) 0 mg / mL Beauveria bassiana toxin, 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v), and (d) 1.2 mg / mL Beauveria bassiana toxin (0.12% w / v), 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v). w / v). Here, % w / v is the percentage w / v of the total volume of the composition, with the remainder being water. [Figure 19] (a) CpGV at 0 μL / L (0% w / v), U+2-ACTX-Hv1a at 0 mg / mL (0% w / v) (control), (b) CpGV at 58.5 μL / L (0.00585% w / v), U+2-ACTX-Hv1a at 0 mg / mL (0% w / v), (c) CpGV at 0 μL / L (0% w / v), U+2-ACTX-Hv1a at 2 mg / mL (0.2% w / v), and (d) CpGV at 58.5 μL / L (0.00585% w / v), U+2-ACTX-Hv1a at 2 mg / mL (0.2% w / v) (where, % The graph shows the mortality rate of hatched codling moss (Cydia pomonella) larvae two days after treatment with w / v (where w / v is the percentage of the total volume of the composition, the remainder being water). [Figure 20]The results of the feed contamination assay with Novaluron and U+2-ACTX-Hv1a are shown, along with a graph evaluating the mortality rate in corn ear worms (Helicoverpa zea) after 3 days. As shown here, there was no evidence that the combination of Novaluron and U+2-ACTX-Hv1a had a greater effect than additive in the corn ear worm (Helicoverpa zea) feed contamination assay. Here, "U+2" refers to U+2-ACTX-Hv1a. The concentrations of Novaluron were as follows: (a) 80 μL / L Novaluron (0.008% w / v), (b) 8 μL / L Novaluron (0.0008% w / v), (c) 0.8 μL / L Novaluron (0.00008% w / v), and (d) 0 μL / L Novaluron (0% w / v). A 10 ppt Spear corresponds to 1 mg / mL (1% w / v) of U+2-ACTX-Hv1a. [Figure 21] The results of the feed contamination assay with nanoparticles and U+2-ACTX-Hv1a are shown, along with a graph evaluating the mortality rate in corn ear worms (Helicoverpa zea) after 3 days. As shown here, there was no evidence that the combination of nanoparticles and U+2-ACTX-Hv1a had a greater effect than the additive effect in the corn ear worm (Helicoverpa zea) feed contamination assay. Here, "Spear" refers to U+2-ACTX-Hv1a. The nanoparticle concentrations were as follows: (a) 50 nm aminated silica (2700 ppm), (b) 50 nm silica (2575 ppm), (c) 20 nm silica (1177 ppm), and (d) 10 nm silica (12500 ppm). Here, "U+2" corresponds to 5 ppt of U+2-ACTX-Hv1a, i.e., 0.5 mg / mL (0.5% w / v of the total volume of the composition) of U+2-ACTX-Hv1a. Proportional mortality = the number of dead insects divided by the total number of insects. "UTC" means the untreated control (water). [Figure 22]The graph shows the dose-response mortality rate of corn ear worms (Helicoverpa zea) after 3 days in a feed contamination assay with cryolite and U+2-ACTX-Hv1a. As shown here, there was no evidence that the combination of cryolite and U+2-ACTX-Hv1a had a greater effect than the additive effect in the corn ear worm (Helicoverpa zea) feed contamination assay. Here, "U+2" refers to U+2-ACTX-Hv1a. The nanoparticle concentrations were as follows: (a) 10000 ppm, (b) 2000 ppm, (c) 400 ppm, and (d) 0 ppm. Here, 10 ppt of "U+2" (i.e., U+2-ACTX-Hv1a) corresponds to 1 mg / mL of U+2-ACTX-Hv1a (1% w / v of the total volume of the composition). Proportional mortality rate = number of dead insects divided by the total number of insects. [Modes for carrying out the invention]

[0033] definition The terms "5' end" and "3' end" refer to directionality, i.e., the orientation of a nucleotide polymer (e.g., DNA) from end to end. The 5' end of a polynucleotide is the end of the polynucleotide that has a carbon at position 5.

[0034] The terms "5' and 3' homology arms," ​​"5' and 3' arms," ​​or "left and right arms" refer to polynucleotide sequences in a vector and / or targeting vector that are recombinant homologously to the target genome sequence and / or endogenous gene in the host organism in order to successfully modify the host organism's chromosomal locus.

[0035] "Γ-CNTX-Pn1a," "γ-CNTX-Pn1a," "gamma-CNTX-Pn1a," or "gamma" refers to an insecticidal neurotoxin derived from the Brazilian armed spider, Phoneutria nigriventer. Γ-CNTX-Pn1a targets the N-methyl-D-aspartate (NMDA) subtype ion channel glutamate receptor (GRIN) and sodium channels.

[0036] "ω / κ-HXTX-Hv1a" or "omega / kappa-HXTX-Hv1a" refers to an insecticidal toxin derived from the Australian Blue Mountain Funnel-web Spider (Hadronyche versuta). ω / κ-HXTX-Hv1a is a type of ACTX peptide, a family of insecticidal ICK peptides isolated from spiders belonging to the Atracinae family. ω / κ-HXTX-Hv1a is a positive allosteric regulator of the nicotinic acetylcholine receptor and also affects insect voltage-gated Ca 2+ Channel and voltage-dependent K + It may be a dual antagonist to the channel. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett. 2019 Jun;593(12):1336-1350, and Windley et al., Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors. Neuropharmacology. 2017 Dec;127:224-242 (these disclosures are incorporated herein by reference in their entirety).

[0037] "ACTX," or "ACTX peptide," or "atrachotoxin" refers to a family of insecticidal ICK peptides isolated from spiders belonging to the family Atracinae. One such spider is known as the Australian Blue Mountains Funnel-web Spider, which has the scientific name Hadronyche versuta. Two examples of ACTX peptides from this species are the omega and U peptides.

[0038] The "ADN1 promoter" refers to a DNA segment consisting of a promoter sequence derived from the Schizosaccharomyces pombe adhesion defect protein 1 gene.

[0039] "Alpha-MF signaling" or "αMF secretory signaling" refers to proteins that guide newly synthesized recombinant polypeptides into the secretory pathway.

[0040] "Agriculturally acceptable carriers" encompass all adjuvants, inert components, dispersants, surfactants, tacks, binders, etc., commonly used in pesticide formulation technology, and these are well known to those skilled in the art of pesticide formulation.

[0041] The term "agriculturally acceptable salt" is used herein as synonymous with the term "pharmaceutically acceptable salt."

[0042] "Agroinfection" refers to a plant transformation method in which DNA is introduced into plant cells using Agrobacteria tumefaciens or Agrobacteria rhizogenes.

[0043] "Alignment" refers to a method of comparing two or more sequences (e.g., nucleotides, polynucleotides, amino acids, peptides, polypeptides, or protein sequences) for the purpose of determining their relationship to each other. Alignment is typically performed by computer programs that apply various algorithms, however, it is also possible to perform alignment manually. Alignment programs typically iterate through the potential alignment of sequences, score the alignments using substitution tables, and employ various strategies to arrive at a potential optimal alignment score. Commonly used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson JD, Higgins DG, Gibson TJ, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22:4673-4680, 1994), CLUSTALV (see Larkin MA, et al., CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics 23(21):2947-2948, 2007), Mafft, Kalign, ProbCons, and T-Coffee (see Notredame et al., T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302:205-217, 2000).Exemplary programs that implement one or more of the algorithms described above include, but are not limited to, MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif. 92121). In some embodiments, alignment involves introducing a "phase shift" and / or "gap" into one or both of the sequences being compared to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relevance of the aligned sequences.

[0044] "Alpha-MF signaling" or "αMF secretory signaling" refers to proteins that guide newly synthesized recombinant polypeptides into the secretory pathway.

[0045] "Arachnida" refers to a class of arthropods. For example, in some embodiments, Arachnida can mean spiders, scorpions, ticks, mites, harvestmen, or sun spiders.

[0046] "Av2," "ATX-II," "neurotoxin 2," "Anemonia viridis toxin 2," or "δ-AITX-Avd1c" refers to a toxin isolated from the venom of Anemonia sulcata. An example of the Av2 polypeptide is the polypeptide with the amino acid sequence of SEQ ID NO: 588.

[0047] "Av3" refers to a polypeptide isolated from the sea anemone Anemonia viridis that can target receptor site 3 on the α subunit III of voltage-gated sodium channels. An example of an Av3 polypeptide is the Av3 polypeptide with the amino acid sequence of SEQ ID NO: 44 (NCBI accession number P01535.1).

[0048] "AVP" or "Av3 variant polypeptide" refers to an Av3 polypeptide sequence and / or polypeptide encoded by a variant Av3 polynucleotide sequence that has been modified to produce a polypeptide and / or polynucleotide sequence that does not exist in nature.

[0049] "BAAS" stands for barley alpha-amylase signal peptide and is an example of an ERSP. An example of BAAS is BAAS (NCBI accession number AAA32925.1) which has the amino acid sequence of SEQ ID NO: 37.

[0050] "Biomass" refers to any measured plant product.

[0051] A "binary vector" or "binary expression vector" refers to an expression vector that can replicate itself in both E. coli and Agrobacterium strains. The vector also contains regions of DNA (often referred to as t-DNA) enclosed by left and right boundary sequences, which are recognized by the pathogenic gene so that they can be copied by Agrobacterium and delivered into plant cells.

[0052] "bp" or "base pair" refers to a molecule containing two chemical bases bonded to each other. For example, the DNA molecule consists of two helical strands, each with a backbone made of alternating deoxyribose and phosphate groups. Each deoxyribose is bonded to one of four bases: adenine (A), cytosine (C), guanine (G), or thymine (T). Adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

[0053] "Bt toxin" refers to the fermentation solids, spores, and toxins produced by Bacillus thuringiensis (Bt)-Gram-positive spore-forming bacteria, such as Bacillus thuringiensis var. kurstaki (Btk), Bacillus thuringiensis var. tenebrionis (Btt), and Bacillus thuringiensis var. israelensis (Bti). During spore formation, Bacillus thuringiensis produces a crystalline protein called δ-endotoxin (i.e., a proteinaceous inclusion body) that has insecticidal properties. In some embodiments, Bt toxin may be a crystalline (Cry) protein, a cytolytic (Cyt) protein, a plant insecticidal protein (Vips), or other toxins produced by Bacillus thuringiensis.

[0054] "Bt-resistant," "Bt-tolerant," "Bt-resistant insect," or "Bacillus thuringiensis toxin-tolerant insect" refers to a genetic change in the susceptibility of a pest population that reflects repeated failures of a product (e.g., Bt) to achieve the expected level of control when used against the pest species.

[0055] The term "C-terminus" refers to the free carboxyl group (i.e., -COOH) located at the end of a polypeptide.

[0056] "cDNA," "copy DNA," or "complementary DNA" refers to a molecule that is complementary to an RNA molecule. In some embodiments, cDNA may be single-stranded or double-stranded. In some embodiments, cDNA may be double-stranded DNA synthesized from a single-stranded RNA template in a reaction catalyzed by reverse transcriptase. In yet other embodiments, "cDNA" refers to all nucleic acids that share the arrangement of sequence elements found in a naturally occurring mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Typically, mRNA species have consecutive exons, and intervening introns are removed by nuclear RNA splicing to produce a continuous open reading frame that codes for proteins. In some embodiments, "cDNA" refers to DNA that is complementary to and derived from an mRNA template.

[0057] "CEW" refers to the corn earworm (Tobacco Budgie).

[0058] For information on "cuttable linkers," please refer to the linker documentation.

[0059] "Cloning" refers to the process and / or method of inserting a DNA segment from one source (e.g., usually a gene of interest, e.g., tvp) and recombining it with a DNA segment from another source (e.g., usually a vector, e.g., plasmid), and instructing the recombinant DNA, or "recombinant DNA," to replicate, usually by transforming a bacterial or yeast host with the recombinant DNA.

[0060] A "chimeric gene" refers to a DNA sequence that codes for a gene derived from a part of one or more coding sequences, thereby producing a new gene.

[0061] A “coding sequence” or “CDS” refers to a polynucleotide or nucleic acid sequence that, under the control of appropriate regulatory sequences and in the presence of necessary transcriptional and / or translational molecular factors, can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) to form a peptide, polypeptide, or protein. The boundaries of a coding sequence are determined by a translation start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. The transcription termination sequence will typically be located on the 3' side of the coding sequence. In some embodiments, the coding sequence may be flanked at the 5' and / or 3' ends by untranslated regions. In some embodiments, the coding sequence can be used to produce peptide, polypeptide, or protein products. In some embodiments, the coding sequence may or may not be fused to another coding sequence or localization signal (e.g., a nuclear localization signal). In some embodiments, the coding sequence may be cloned into a vector or expression construct, incorporated into a genome, or exist as a DNA fragment.

[0062] "Codon optimization" refers to the production of genes in which one or more endogenous, native, and / or wild-type codons are ultimately replaced with preferred codons in the corresponding host, although they still ultimately code for the same amino acid.

[0063] "Combination" means any association between two or more items. The association may be spatial, temporal, and / or may refer to the use of two or more items for a common purpose. For example, a combination may be any spatiotemporal association, mixture, or permutation of (1) one or more CRIP or pharmaceutically acceptable salts thereof, CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) described herein, and (1) and (2) are used for the common purpose of controlling or eliminating insect pests such that the insect pests die, stop or slow their movement, stop or slow their feeding, stop or slow their growth, are confused (e.g., with respect to navigation, food location, sleep behavior, and / or mating), fail to pupate, are prevented from reproducing, and / or are prevented from producing offspring, and / or are prevented from producing fertile offspring.

[0064] Unless otherwise specified in the context, the term “combination” may include (1) administering one or more CRIP or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, together with (2) one or more insecticides (IA), in any order, simultaneously, separately, or sequentially.

[0065] In some embodiments, it will be understood that (1) one or more CRIP or pharmaceutically acceptable salts thereof, CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) are considered to be administered as a “combination” or “in combination” if the pest, or the site to which the pest is exposed, or the site to which the pest is protected from the pest (e.g., a plant), is treated with simultaneous exposure to both (1) and (2). In some embodiments, each of (1) one or more CRIP or pharmaceutically acceptable salts thereof, CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) may be administered sequentially or according to different schedules, and the individual doses of different agents do not need to be administered simultaneously or in the same composition. Rather, insofar as (1) one or more CRIP or pharmaceutically acceptable salts thereof, CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) remain pesticide-effective (i.e., possess insecticidal activity), they are considered to be administered “in combination.”

[0066] In some embodiments, “combination” means administering (1) one or more CRIP or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or a combination thereof, simultaneously with (2) one or more insecticides (IA).

[0067] In some embodiments, “combination” means administering (1) one or more CRIP or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, separately with (2) one or more insecticides (IA).

[0068] In further embodiments, “combination” means administering (1) one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, in any order, in sequence with (2) one or more insecticides (IA).

[0069] In some embodiments of the present invention, i.e., when the combination is administered sequentially or separately, the delay in administering the second component should not result in a loss of the beneficial effect of the overall combination (i.e., a combination of (1) one or more CRIP or pharmaceutically acceptable salts thereof, one or more CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or a combination thereof, and (2) one or more insecticides (IA)). When a combination of two or more components is administered separately or sequentially, it will be understood that the dosing regimen for each component may be different from and independent of the other components.

[0070] In some embodiments, one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or a combination thereof may be administered on the same day as one or more insecticides (IA). In other embodiments, one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or a combination thereof may be administered in the same week or month as one or more insecticides (IA).

[0071] In some embodiments, the combination may be a “mixture.” As used herein, “mixture” means a combination of two or more agents, for example, (1) one or more CRIP or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or a combination thereof, in physical and / or chemical contact with (2) one or more insecticides (IA).

[0072] "Complementary," as understood by those skilled in the art, refers to the topological compatibility or agreement of the interaction surfaces of two polynucleotides. Thus, two sequences are "complementary" if they can hybridize to each other to form a stable antiparallel double-stranded nucleic acid structure. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under strict hybridization conditions. Therefore, a polynucleotide with the sequence 5'-TATAC-3' is complementary to a polynucleotide with the sequence 5'-GTATA-3'.

[0073] "Conditioned culture medium" refers to a cell culture medium that is used by cells and is concentrated with cell-derived substances, but does not contain cells themselves.

[0074] "Cone shell," "cone snail," or "cone" refers to organisms belonging to the genus Conus of predatory marine gastropods. For example, in some embodiments, a cone shell may be one of the following species: Conus amadis, Conus catus, Conus ermineus, Conus geographus, Conus gloriamaris, Conus kinoshitai, Conus magus, Conus marmoreus, Conus purpurascens, Conus stercusmuscarum, Conus striatus, Conus textile, or Conus tulipa.

[0075] "Conotoxin" refers to a toxin isolated from the cone snail that acts by interfering with neuronal communication. For example, in some embodiments, the conotoxin may be α-, ω-, μ-, δ-, or κ-conotoxin. Briefly, α-conotoxin (and αA- and φ-conotoxins) target nicotinic ligand-gated channels, ω-conotoxin targets voltage-gated calcium channels, μ-conotoxin targets voltage-gated sodium channels, δ-conotoxin targets voltage-gated sodium channels, and κ-conotoxin targets voltage-gated potassium channels.

[0076] "Copy number" refers to the number of identical copies of a vector, expression cassette, amplification unit, gene, or any defined nucleotide sequence that are present in a host cell at any given time. For example, in some embodiments, a gene or another defined chromosomal nucleotide sequence may exist in one, two, or more copies on a chromosome. An autonomous replicating vector may exist in one or several hundred copies per host cell.

[0077] "CRIP" refers to a cysteine-rich insecticidal peptide. CRIP is a peptide rich in cysteine ​​residues, and in some embodiments, it is operable to form disulfide bonds between such cysteine ​​residues. In some embodiments, CRIP contains at least four, sometimes six, and sometimes eight cysteine ​​amino acids in a protein or peptide having at least 10 amino acids, and the cysteine ​​forms two, three, or four disulfide bonds. In some embodiments, the disulfide bonds contribute to the folding, three-dimensional structure, and activity of the insecticidal peptide. The cysteine-cysteine ​​disulfide bonds and the three-dimensional structure they form play an important role in the insecticidal properties of these insecticidal peptides. In some embodiments, CRIP may or may not contain an inhibitor-cystine knot (ICK) motif. For example, in some embodiments, the CRIP having an ICK motif may be an ACTX peptide derived from spiders, and in other embodiments, the CRIP without an ICK motif, i.e., non-ICK CRIP, may be peptides such as Av2 and Av3 isolated from sea anemones. Non-ICK CRIPs may have 4 to 8 cysteine ​​molecules forming 2 to 4 disulfide bonds. These cysteine-cysteine ​​disulfide bond-stabilized toxic peptides (CRIPs) can exhibit remarkable stability when exposed to the environment. Many CRIPs have been isolated from venomous animals such as spiders, scorpions, snakes, seashells, and sea anemones, and they are toxic to insects.

[0078] "CRIP construct" refers to the three-dimensional arrangement / orientation of operably linked polypeptide segments (e.g., CRIP-insecticidal protein) of peptides, polypeptides, and / or motifs. For example, a CRIP expression ORF can include one or more of CRIP, endoplasmic reticulum signal peptide (ERSP), linker peptide (L), translation stabilization protein (STA), or any combination thereof. As used herein, the term "CRIP construct" is used to describe the designation and / or orientation of a structural motif. In other words, a CRIP construct describes the arrangement and orientation of components or motifs contained within a given CRIP expression ORF. For example, in some embodiments, the CRIP construct describes the orientation of one of the following CRIP-insecticidal proteins, without limitation: ERSP-CRIP, ERSP-(CRIP) N , ERSP-CRIP-L, ERSP-(CRIP) N -L, ERSP-(CRIP-L) N , ERSP-L-CRIP, ERSP-L-(CRIP) N , ERSP-(L-CRIP) N , ERSP-STA-CRIP, ERSP-STA-(CRIP) N , ERSP-CRIP-STA, ERSP-(CRIP) N -STA, ERSP-(STA-CRIP) N , ERSP-(CRIP-STA) N , ERSP-L-CRIP-STA, ERSP-L-STA-CRIP, ERSP-L-(CRIP-STA) N , ERSP-L-(STA-CRIP) N , ERSP-L-(CRIP) N -STA, ERSP-(L-CRIP) N -STA, ERSP-(L-STA-CRIP) N , ERSP-(L-CRIP-STA) N , ERSP-(L-STA) N -CRIP, ERSP-(L-CRIP)<s N -STA, ERSP-STA-L-CRIP, ERSP-STA-CRIP-L, ERSP-STA-L-(CRIP)N ERSP-(STA-L) N -CRIP, ERS-STA-(L-CRIP) N ERSP-(STA-L-CRIP) N ERSP-STA-(CRIP) N -L, ERSP-STA-(CRIP-L) N ERSP-(STA-CRIP) N -L, ERSP-(STA-CRIP-L) N , ERSP-CRIP-L-STA, ERSP-CRIP-STA-L, ERSP-(CRIP) N -STA-L, ERSP-(CRIP-L) N -STA, ERSP-(CRIP-STA) N -L, ERSP-(CRIP-L-STA) N , or ERSP-(CRIP-STA-L) N (Here, N is an integer in the range of 1 to 200). See also "Structural Motifs".

[0079] A "CRIP ORF diagram" refers to a composition of one or more CRIP ORFs described in diagrammatic or formulaic form. For example, a "CRIP ORF diagram" may be described using initials or abbreviations (short-hand references) for the DNA segments contained within the ORF. Thus, in one embodiment, a "CRIP ORF diagram" can describe the polynucleotide segments encoding ERSP, L, STA, and CRIP by illustrating the DNA segments in formulaic form as "ersp" (i.e., a polynucleotide sequence encoding the ERSP polypeptide), "linker" or "L" (i.e., a polynucleotide sequence encoding the LINKER polypeptide), "sta" (i.e., a polynucleotide sequence encoding the STA polypeptide), and "crip" (i.e., a polynucleotide sequence encoding CRIP). An example of a CRIP ORF diagram is "ersp-sta-(linker i -crip j ) N " or "ersp-(cripj - Linker i ) N -sta" and / or any combination of those DNA segments.

[0080] "CRIP polynucleotide" refers to a polynucleotide or group of polynucleotides capable of expressing and / or encoding an insecticidal protein that contains one or more CRIP polypeptides or proteins in addition to one or more CRIP polypeptides.

[0081] "CRIP-insecticide protein" refers to any protein, peptide, polypeptide, amino acid sequence, composition, or arrangement consisting of: (1) at least one CRIP, or two or more CRIPs, and (2) additional peptides, polypeptides, or proteins, wherein the additional peptides, polypeptides, or proteins have the ability to: (a) increase mortality and / or inhibit insect growth when insects are exposed to the CRIP-insecticide protein, compared to CRIP alone; (b) increase the expression of the CRIP-insecticide protein, for example, in host cells or expression systems; and / or (c) affect the post-translational processing of the CRIP-insecticide protein.

[0082] In some embodiments, the insecticidal protein may contain one or more CRIPs disclosed herein. In some embodiments, the CRIP-insecticidal protein may be a polymer containing two or more CRIPs. In some embodiments, the insecticidal protein may contain a CRIP homopolymer (e.g., two or more CRIP monomers having the same CRIP). In some embodiments, the insecticidal protein may contain a CRIP heteropolymer (e.g., two or more CRIP monomers having different CRIP monomers).

[0083] In some embodiments, the CRIP-insecticidal protein may be a polymer of amino acids that exhibits insecticidal activity against one or more insect species when properly folded or in its most natural thermodynamic state.

[0084] In some embodiments, the CRIP-insecticide protein may be a polymer containing two or more CRIPs, where the CRIPs are operably linked via linker peptides (e.g., cleavable and / or non-cleavable linkers). In some embodiments, the CRIP-insecticide protein may refer to one or more CRIPs operably linked to one or more proteins such as a stabilizing domain (STA), an endoplasmic reticulum signaling protein (ERSP), an insect-cleavable or insect-non-cleavable linker (L), and / or any other combination thereof. In some embodiments, the CRIP-insecticide protein may be (1) a wild-type CRIP protein, and (2) a non-naturally occurring protein containing additional peptides, polypeptides, or proteins (e.g., ERSP, linker, STA, UBI, or histidine tag or similar marker).

[0085] "Culture" or "cell culture" refers to the maintenance of cells in an artificial in vitro environment.

[0086] "Culture" refers to the growth of organisms on or in various types of culture media. For example, the term "cultivation" can mean the growth of a cell population under favorable conditions in a liquid or solid medium. In some embodiments, cultivation refers to the fermentative recombinant production of a desired heterologous polypeptide and / or other desired end product (typically in a container or reactor).

[0087] "Cystine" refers to an oxidized cysteine ​​dimer. Cystine is a sulfur-containing amino acid obtained through the oxidation of two cysteine ​​molecules, linked by a disulfide bond.

[0088] A "standard culture medium" refers to a culture medium that consists of known chemical components but does not contain by-products such as crude protein extracts, yeast extracts, or peptones.

[0089] "Degeneracy" or "codon degeneracy" refers to the phenomenon in which a single amino acid can be encoded by different nucleotide codons. Therefore, the nucleic acid sequence of a nucleic acid molecule encoding a protein or polypeptide can be altered by degeneracy. As a result of genetic coding degeneracy, many nucleic acid sequences can encode a given polypeptide with specific activity, and such functionally equivalent variants are intended herein.

[0090] "Desmethyllimosin B" refers to [(5R,7R,8R,9R,10R,13S,17S)-17-[(3R)-5-hydroxyoxolan-3-yl]-4,4,8,10,13-pentamethyl-3,16-dioxo-6,7,9,11,12,17-hexahydro-5H-cyclopenta[a]phenanthren-7-yl]acetate.

[0091] A "disulfide bond" refers to a covalent bond between two cysteine ​​amino acids that is induced by the coupling of two thiol groups on the side chain.

[0092] "DNA" refers to deoxyribonucleic acid, which comprises a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]) that can be arranged in single-stranded or double-stranded forms. For example, one or more nucleotides can form a polynucleotide.

[0093] "dNTP" refers to nucleoside triphosphates, which are components of DNA and RNA.

[0094] A "dual expression cassette" refers to an expression cassette containing two different polypeptides on the same vector.

[0095] A "dual transgene peptide expression vector" or "dual transgene expression vector" refers to a yeast expression vector containing two copies of a heterologous polypeptide expression cassette.

[0096] "Endogenous" refers to polynucleotides, peptides, polypeptides, proteins, or processes that are naturally occurring and / or present in living organisms, such as molecules or activities that are already present in the host cell prior to a particular genetic manipulation.

[0097] An "enhancer element" refers to a DNA sequence operably ligated to a promoter, which can exert increased transcriptional activity on the promoter compared to the transcriptional activity produced by the promoter in the absence of the enhancer element.

[0098] The endoplasmic reticulum (ER), or "ER," is an intracellular organelle common to all eukaryotes, where several post-translational modification processes occur.

[0099] "ERSP," or "endoplasmic reticulum signal peptide," is the N-terminal sequence of amino acids that is recognized and bound by a host cell signal recognition particle during protein translation of the mRNA molecule encoding CRIP. This causes the ribosome / mRNA complex of protein translation to move to the ER in the cytoplasm. As a result, protein translation pauses until it docks with the ER, where it continues and the resulting protein is injected into the ER.

[0100] "ersp" refers to the polynucleotide that codes for the peptide ERSP.

[0101] "ER transport" refers to the transport of proteins expressed in cells to the ER for post-translational modification, sorting, and transport.

[0102] "Excipients" refers to any pharmacologically inactive, natural or synthetic components or substances formulated together with (e.g., simultaneously with) or subsequently with the active ingredient of the present invention (i.e., CRIP or CRIP-insecticidal protein). In some embodiments, excipients may be any additives, adjuvants, binders, fillers, carriers, coatings, diluents, disintegrants, fillers, lubricants, preservatives, vehicles, or combinations thereof that can be administered together with CRIP or CRIP-insecticidal protein of the present invention and / or are useful in the preparation of the compositions of the present invention. Excipients include any such materials known in the art that are non-toxic and do not interact with other components of the composition. In some embodiments, excipients may be formulated together with CRIP or CRIP-insecticidal protein when preparing a composition for the purpose of increasing its volume (and therefore often referred to as fillers, fillers, or diluents). In other embodiments, excipients may be used to enhance the active ingredient in the final dosage form, such as by promoting absorption and / or solubility. In further embodiments, excipients may be used to provide stability or to prevent contamination (e.g., microbial contamination). In other embodiments, excipients may be used to impart physical properties to a composition (e.g., a composition in the physical form of dry granules or dry flowable powder). References to excipients include both one and more such excipients. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, by EW Martin (the disclosure thereof is incorporated herein by reference in its entirety).

[0103] An "expression cassette" refers to (1) a target DNA sequence (e.g., a polynucleotide capable of encoding CRIP) and one or more of the following: (2) a promoter, terminator, and / or enhancer element; (3) a suitable mRNA-stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) an intron; and / or (6) a post-transcriptional regulatory element. The combination of (1) and at least one of (2) to (6) is referred to as an "expression cassette." In some embodiments, there may be multiple expression cassettes cloned into the vector. For example, in some embodiments, there may be a first expression cassette containing a polynucleotide capable of encoding CRIP. In alternative embodiments, there may be two expression cassettes (i.e., a dual expression cassette), each containing a polynucleotide capable of encoding CRIP. In other embodiments, there may be three expression cassettes (i.e., a triple expression cassette) capable of encoding CRIP. In some embodiments, a dual expression cassette may be produced by subcloning a second expression cassette into a vector containing a first expression cassette. In some embodiments, a triple expression cassette may be produced by subcloning a third expression cassette into a vector containing a first expression cassette and a second expression cassette. Methods relating to expression cassettes and cloning techniques are well known in the art and are described herein. See also CRIP expression cassettes.

[0104] "Expressed ORF" refers to the nucleotides that encode the protein complex, and is defined as the nucleotides of the ORF.

[0105] "FECT" refers to a transient plant expression system using foxtail mosaic virus with the coating protein gene and triple gene block eliminated.

[0106] "Fermentation beer" refers to the used fermentation medium (i.e., the supernatant of the fermentation medium after removal of organisms) that has been inoculated into and consumed by transformed host cells (e.g., yeast cells capable of expressing CRIP of the present invention). In some embodiments, fermentation beer refers to the solution recovered after fermentation of the transformed host cells. The term "fermentation" broadly refers to the enzymatic and anaerobic or aerobic degradation of organic matter (e.g., carbon substrates) by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, etc.) to produce fermentation products (e.g., one or more peptides of the present invention). While fermentation, as used herein, typically describes a process occurring under anaerobic conditions, the term "fermentation" as used herein may also occur in the presence of oxygen, and is not intended to limit the term to strictly anaerobic conditions.

[0107] "Fermentation solids" refer to the solids (including dissolved ones) that remain in the fermented beer during the yeast-based fermentation process, and essentially consist of salts with a molecular weight cutoff of approximately 200 kDa to 1 kDa, complex protein sources, vitamins, and additional yeast by-products.

[0108] "GFP" stands for green fluorescent protein from the jellyfish, Aequorea victoria.

[0109] "HIS" or "His" refers to histidine. For example, in some embodiments, "HIS" or "His" may refer to a histidine tag (e.g., a histidine tag having the amino acid sequence shown in SEQ ID NO: 591).

[0110] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If the positions in both of the two sequences being compared are occupied by the same base or amino acid monomer subunit (for example, if the positions in each of the two DNA molecules are occupied by adenine), then these molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Therefore, in some embodiments, the term "homologous" refers to sequence similarity between two polypeptide molecules or two nucleic acid molecules. If the positions in both of the two sequences being compared are occupied by the same base or amino acid monomer subunit (for example, if the positions in each of the two DNA molecules are occupied by adenine), then these molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, two sequences are 60% homologous if 6 out of 10 positions are identical or homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology.

[0111] When used in relation to nucleic acids, the term "homology" refers to the degree of complementarity. Partial homology or complete homology (and therefore identical) can exist. "Sequence identity" refers to a measure of the relevance between two or more nucleic acids, given as a percentage with respect to the total comparison length. The calculation of identity takes into account those nucleotide residues that are identical and in the same relative position in each larger sequence.

[0112] "Homologous recombination" refers to the event of replacing a DNA segment with another segment that has an identical (homologous) or nearly identical region. For example, in some embodiments, "homologous recombination" refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. In short, homologous recombination is most widely used by cells to precisely repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Although homologous recombination varies considerably between different organisms and cell types, most forms involve the same basic steps: after a double-strand break occurs, a process called excision cuts a section of DNA around the 5' end of the break. Subsequently, in a strand entry step, the protruding 3' end of the broken DNA molecule then "enters" an unbroken, similar or identical DNA molecule. After strand entry, further sequences of the event may follow one of two main pathways: the double-strand break repair pathway or the synthesis-dependent strand annealing pathway. Homologous recombination is conserved across all three domains of organisms and all viruses, suggesting that it is a nearly universal biological mechanism. For example, in some embodiments, homologous recombination may occur using site-directed integration (SSI) sequences, thereby resulting in strand-swapping cross-events between substantially similar nucleic acid sequences in a nucleotide composition. These cross-events can occur between sequences contained in the targeted construct of the present invention (i.e., SSI sequences) and endogenous genomic nucleic acid sequences (e.g., polynucleotides encoding peptide subunits). Furthermore, in some embodiments, two or more site-directed homologous recombination events may occur, resulting in substitution events in which nucleic acid sequences contained within the targeted construct replace specific sequences present in the endogenous genomic sequence.

[0113] An "ICK motif," "ICK motif protein," "inhibitor cystine knot motif," "ICK peptide," "cystine knot motif," or "cystine knot peptide" refers to a 16-60 amino acid peptide having at least six half-cystine core amino acids with three disulfide crosslinks, where the three disulfide crosslinks are covalent, and the covalent disulfide bonds among the six half-cystine residues are located between the 1st and 4th, 2nd and 5th, and 3rd and 6th half-cystines of the six half-cystine core amino acids starting from the N-terminal amino acid. Generally, this type of peptide typically contains a beta-hairpin secondary structure consisting of a residue located between the 4th and 6th core half-cystine of the motif, and the hairpin is stabilized by a structural crosslink provided by the three disulfide bonds of the motif. Note that additional cysteine / cystine or half-cystine amino acids may be present within the inhibitor cystine knot motif.

[0114] "ick" refers to the nucleotide that codes for the ICK motif protein.

[0115] "ICK motif protein expression ORF" or "expression ORF" refers to the nucleotides that encode the ICK motif protein complex, and is defined as nucleotides in an ORF.

[0116] An "ICK motif protein expression vector," or "ICK expression vector," or "ICK motif expression vector" means a binary vector containing an expression ORF. The binary vector also includes the necessary transcription promoter and terminator sequences around the expression ORF to promote the expression of the ORF and the protein it encodes.

[0117] "Identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, which is determined by comparing the sequences. The term "identity" may also, in some cases, mean the degree of sequence relevance between polypeptide sequences or polynucleotide sequences, determined by the matching of strings of such sequences. "Identity" and "similarity" can be readily calculated by any one of the countless methods known to those skilled in the art, as described below: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied This includes, but is not limited to, Math., 48:1073 (1988) (these disclosures are incorporated herein by reference in their entirety). Furthermore, methods for determining identity and similarity are codified in publicly available computer programs.For example, in some embodiments, methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. et al., J. Molec. Biol. 215:403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al. NCBI, NLM NIH Bethesda, Md20894; Altschul, S., et al. J. Mol. Biol. 215:403-410 (1990)) (these disclosures are incorporated herein by reference in their entirety).

[0118] "IGER" stands for Short Peptide Name, based on an actual sequence of single-letter codes. This is an example of an intervening linker.

[0119] "In vivo" refers to the natural environment (e.g., animals or cells), and processes or reactions that occur within the natural environment.

[0120] "Inactive" refers to a state in which something is not in a state of use, for example, dormant and / or non-functional. For example, when used in the context of genes or referred to in relation to genes, the term inactive means that the gene no longer actively synthesizes a gene product, does not translate the gene product into a protein, or otherwise prevents the gene from performing its normal function. For example, in some embodiments, the term inactive can refer to a gene's failure to transcribe RNA, failure of RNA processing (e.g., premRNA processing, RNA splicing, or other post-transcriptional modifications), interference with non-coding RNA maturation, interference with RNA transport (e.g., from nucleus to cytoplasm), interference with translation, protein folding, translocation, protein transport, and / or inhibition and / or interference with any of the molecular polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors contributing to any of the aforementioned processes.

[0121] "Inoperable" refers to a state in which a gene is not functioning, malfunctioning, or no longer able to function. For example, when used in the context of a gene, or when referring to a gene, the term "inoperable" means that the gene is no longer able to function as usual, either permanently or transiently. For example, in some embodiments, "inoperable" means that the gene is no longer able to synthesize a gene product, does not translate the gene product into a protein, or is otherwise unable to perform its normal function. For example, in some embodiments, the term "inoperable" may refer to a gene's failure to transcribe RNA, failure of RNA processing (e.g., premRNA processing, RNA splicing, or other post-transcriptional modifications), interference with non-coding RNA maturation, interference with RNA transport (e.g., from nucleus to cytoplasm), interference with translation, protein folding, translocation, protein transport, and / or inhibition and / or interference with any of the molecular polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors contributing to any of the aforementioned processes.

[0122] The term "insect" includes all organisms of the class "Insecta". The term "pre-adult" refers to any form of an organism prior to adulthood, including, for example, eggs, larvae, and nymphs. As used herein, the term "insect" refers to any arthropod and nematode, including mites, and all insects known to infest crops, vegetables, and trees, including insects considered pests in the fields of forestry, horticulture, and agriculture. Examples of specific crops that may be protected in the manner disclosed herein are soybeans, corn, cotton, alfalfa, and vegetable crops. A list of specific crops and insects is provided here.

[0123] "Insect intestinal environment" or "intestinal environment" refers to the specific pH and proteinase conditions found in the foregut, midgut, or hindgut of insects or insect larvae.

[0124] "Insect hemolymphatic environment" refers to specific pH and proteinase conditions found within insects or insect larvae.

[0125] "Insecticidal activity" means that, upon or after exposure of an insect to a compound, substance, or peptide, the insect dies, stops or slows its movement, stops or slows its feeding, stops or slows its growth, is confused (e.g., with respect to navigation, food location, sleep behavior, and / or mating), fails to pupate, is prevented from reproducing, and / or prevents the insect from producing offspring, and / or prevents the insect from producing fertile offspring.

[0126] "Insecticide," "IA," or "drug" means one or more chemical substances, molecules, nucleotides, polynucleotides, RNA, DNA, peptides, polypeptides, proteins, lipids, glycolipids, enzymes, toxins, toxic substances, poisons, insecticides, pesticides, organic compounds, inorganic compounds, viruses, prokaryotes, or eukaryotes (and drugs produced from such prokaryotes or eukaryotes). In some embodiments, IA includes RNAi, gastrotoxicants, type O chitin biosynthesis inhibitors, type I chitin biosynthesis inhibitors, insect viruses, and Azadirachta. Compounds isolated from indica, compounds with unknown MOA, bacteria (and their products), fungi (and their products), nematodes (and their products), plant extracts, mechanical disruptors, fluorescent whitening agents, silica nanospheres, chitinases, lectins, membrane invasion complex / perforin (MACPF) proteins, plant virus coat protein-toxin fusions, glycan-binding domain / toxin fusion proteins, acetylcholinesterase (AchE) inhibitors, GABAergic chloride channel blockers, sodium channel modulators, nicotinic acetylcholine receptor (nAchR) competitive modulators, nicotinic acetylcholine receptor (nAchR) allosteric modulators - site I, glutamatergic chloride channel (GluCl) allosteric modulators, juvenile hormone mimetic compounds, and other nonspecific compounds. This includes, but is not limited to, members selected from the categories of target (multisite) inhibitors, chordominant organ TRPV channel modulators, mite growth inhibitors, mitochondrial ATP synthase inhibitors, oxidative phosphorylation uncouplers via proton gradient disruption, nicotinic acetylcholine receptor (nAchR) channel blockers, molting disruptors (diptera), Edison receptor agonists, octopamine receptor agonists, mitochondrial complex III electron transport inhibitors, mitochondrial complex I electron transport inhibitors, voltage-gated sodium channel blockers, acetyl-CoA carboxylase inhibitors, mitochondrial complex IV electron transport inhibitors, mitochondrial complex II electron transport inhibitors, ryanodine receptor modulators, chordominant organ modulators - undefined target sites, or GABAergic chloride channel allosteric modulators.In some embodiments, the insecticide may be a polymer of amino acids, peptides, polypeptides, or proteins, and such peptide-IA may be prepared and / or used according to any of the peptide and / or protein methods described herein.

[0127] An "integrated expression vector" or "integrated vector" refers to a yeast expression vector that can be inserted into a specific gene locus in the yeast cell genome and stably become part of the yeast genome.

[0128] "Insecticide-resistant," "insecticide-resistant," "insecticide-resistant insect," or "insecticide-resistant insect" refers to a genetic change in the susceptibility of a pest population to an insecticide that reflects repeated failures of the insecticide to achieve the expected level of control when used against the pest species.

[0129] An "intermediate linker" refers to a short peptide sequence in a protein that separates different parts of the protein, or a short DNA sequence placed in a reading frame in an ORF to separate upstream and downstream DNA sequences. For example, in some embodiments, intermediate linkers can be used to allow a protein to achieve the formation of independent secondary and tertiary structures during translation. In some embodiments, intermediate linkers may be either resistant to or susceptible to cleavage in plant cell environments, insect and / or Lepidopteran intestinal environments, and insect hemolymph and Lepidopteran hemolymph environments.

[0130] "Isolated" refers to separating a substance and / or component from its natural environment. For example, a toxin isolated from a given genus or species means that the toxin has been isolated from its natural environment (e.g., extracted from a wild-type organism).

[0131] "Kappa-ACTX peptide" refers to an excitatory toxin that inhibits insect calcium-activated potassium (KCa) channels (Slo type). As used herein, "Kappa-ACTX peptide" may refer to peptides isolated from the Australian blue mountain funnel-web spider, Hadronyche versuta, or their variants.

[0132] "kb" refers to a kilobase (i.e., 1,000 bases). As used herein, the term "kb" means the length of a nucleic acid molecule. For example, 1 kb refers to a nucleic acid molecule that is 1,000 nucleotides long. A 1 kb length double-stranded DNA molecule contains 2,000 nucleotides (i.e., 1,000 nucleotides per strand). Alternatively, a 1 kb length single-stranded RNA molecule contains 1,000 nucleotides.

[0133] "kDa" refers to a kilodalton (a unit equivalent to 1,000 daltons), while "dalton" or "Da" is a unit of molecular weight (MW).

[0134] "Knock-in," "knock-in," "knocks-in," or "knocking-in" refers to replacing an endogenous gene with an exogenous or heterologous gene or a portion thereof. For example, in some embodiments, the term "knock-in" refers to introducing a nucleic acid sequence encoding a desired protein into a target locus by homologous recombination, thereby causing the expression of the desired protein. In some embodiments, a "knock-in" mutation can be a modification of a gene sequence to produce a loss-of-function or gain-of-function mutation. The term "knock-in" can refer to a procedure in which an exogenous or heterologous polynucleotide sequence or fragment thereof is introduced into the genome (e.g., "they performed a knock-in," or "they knocked in a heterologous gene"), or to the resulting cell and / or organism (e.g., "the cell is knock-in," or "the animal is knock-in").

[0135] "Knock out," "knockout," "knock-out," "knock-out," "knock-out," or "knocking-out" refers to the partial or complete suppression of the expression gene product (e.g., mRNA) of a protein encoded by the cell's endogenous DNA sequence. In some embodiments, "knockout" may be achieved by targeted deletion of an entire gene or a portion of a gene encoding a peptide, polypeptide, or protein. As a result, the deletion can inactivate, partially inactivate, disable, partially disable, or otherwise reduce the expression of the gene or its product in any cell of an organism and / or cell that normally expresses the gene. The term "knockout" may refer to a procedure in which an endogenous gene is completely or partially inactivated or disabled (e.g., "they performed a knockout," or "they knocked out an endogenous gene"), or to the resulting cell and / or organism (e.g., "the cell is knockout," or "the animal is knockout").

[0136] "Knockdown dose 50" or "KD 50 " refers to the median dose required to cause paralysis or cessation of movement in 50% of a population (e.g., a population of Musca domestica (houseflies) and / or Aedes aegypti (mosquitoes)).

[0137] "l" or "linker" refers to the nucleotide that codes for the linker peptide.

[0138] In the appropriate context, "L" refers to a linker peptide that ligates a translation-stabilizing protein (STA) to additional polypeptides (e.g., heterologous peptides and / or multiple heterologous peptides). When referring to amino acids, "L" can also mean leucine.

[0139] The "LAC4 promoter" or "Lac4 promoter" refers to a DNA segment consisting of a promoter sequence derived from the K. lactis β-galactosidase gene. The LAC4 promoter is a potent, inducible reporter used to drive the expression of transformed exogenous genes in yeast.

[0140] The term "LAC4 terminator" or "Lac4 terminator" refers to a DNA segment consisting of a transcriptional terminator sequence derived from the K. lactis β-galactosidase gene.

[0141] "LD 20 " refers to the dose required to kill 20% of the population.

[0142] "LD 50 "50" refers to the lethal dose, which is the amount needed to kill 50% of a population.

[0143] The term "Lepidopteran gut environment" refers to the specific pH and proteinase conditions found in the foregut, midgut, or hindgut of Lepidopteran insects or larvae.

[0144] The term "Lepidopteran hemolymphatic environment" refers to specific pH and proteinase conditions found within Lepidopteran insects or larvae.

[0145] "Linker," "peptide linker," "L," or "intermediate linker" refers to a short peptide sequence capable of acting to link two peptides together. A linker may refer to a short DNA sequence placed within the reading frame of an ORF to separate upstream and downstream DNA sequences. In some embodiments, the linker may be cleavable by an insect protease. In some embodiments, the linker may allow the protein to achieve the formation of independent secondary and tertiary structures during translation. In some embodiments, the linker may be resistant or sensitive to cleavage in the plant cell environment, the insect and / or Lepidopteran intestinal environment, and / or the insect hemolymph and Lepidopteran hemolymph environment. In some embodiments, the linker may be cleaved by a protease. For example, in some embodiments, the linker may be cleaved by plant proteases (e.g., papain, bromelan, ficin, actinidine, zingibain, and / or cardosin), insect proteases, fungal proteases, vertebrate proteases, invertebrate proteases, bacterial proteases, mammalian proteases, reptile proteases, or avian proteases. In some embodiments, the linker may be cleavable or incleavable. In some embodiments, the linker may include a secondary or tertiary region, each region of which is cleavable by at least two types of proteases, one of which is an insect and / or nematode protease and the other is a human protease. In some embodiments, the linker may have one of (at least) three roles: being cleaved in the insect intestinal environment, being cleaved in plant cells, or being designed not to be cleaved.

[0146] "Medium" (plural: "media") refers to the nutrient solution used to culture cells in cell culture.

[0147] "MOA" refers to the mechanism of action.

[0148] "Molecular weight (MW)" refers to the mass or weight of a molecule and is typically measured in "Daltons (Da)" or "kilodaltons (kDa)". In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering. In some embodiments, the SDS-PAGE method is as follows: The sample of interest is separated on a gel using a set of molecular weight standards. The sample is electrophoresed, then the gel is treated with the desired stain, followed by destaining for about 2 to 14 hours. The next step is to determine the relative migration distance (Rf) between the standards and the protein of interest. The migration distance can be determined using the following formula:

number

[0149] Next, the logarithm of MW can be determined based on the values ​​obtained for the band in the standard, for example, in some embodiments, the logarithm of the molecular weight of the SDS-modified polypeptide and its relative migration distance (Rf) are plotted on a graph. After plotting the graph, the derived values ​​can be interpolated to obtain the molecular weight of the unknown protein band.

[0150] A "motif" refers to a polynucleotide sequence or polypeptide sequence that is involved in having some biological significance and / or exerting some effect or participating in some biological process.

[0151] A "multiplex cloning site" or "MCS" refers to a segment of DNA found on a vector that contains multiple restriction sites into which the target DNA sequence can be inserted.

[0152] A “mutant” means an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein that has a change or variation (for example, in a nucleotide sequence or amino acid sequence) so that the organism and / or sequence differs from a naturally occurring organism or wild-type organism, wild-type sequence, and / or reference sequence compared to the mutant. In some embodiments, this change or variation may be a substitution or modification (e.g., deletion or addition) of one or more nucleotides and / or amino acids. In some embodiments, one or more amino acid substitutions or modifications may be conserved. Here, such conserved amino acid substitutions and / or modifications in the “mutant” do not substantially reduce the activity of the mutant in relation to the non-mutant form of the mutant. For example, in some embodiments, the “mutant” has one or more conserved amino acid substitutions compared to a peptide having the disclosed sequence and / or claimed sequence, as indicated by the sequence number.

[0153] The "N-terminus" refers to the free amine group (i.e., -NH2) located at the beginning or start of a polypeptide.

[0154] "NCBI" refers to the National Center for Biotechnology Information.

[0155] "nm" refers to nanometers.

[0156] "Non-ICK CRIP" refers to peptides containing 4 to 8 cysteine ​​molecules that form 2 to 4 disulfide bonds. Non-ICK peptides include cystine knot peptides that are not ICK peptides. Non-ICK peptides may have a different disulfide bond binding pattern than ICK peptides. Examples of non-ICK CRIPs include peptides such as Av2 and Av3 isolated from sea anemones, and these sea anemone peptides are examples of a class of compounds that regulate sodium channels in the peripheral nervous system (PNS) of insects.

[0157] "Nonpolar amino acids" are weakly hydrophobic amino acids, including glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine, and methionine. Glycine or gly is the most preferred nonpolar amino acid for the dipeptide of the present invention.

[0158] "Normalized peptide yield" refers to the peptide yield in the culture medium divided by the corresponding cell density at the time the peptide yield was measured. Peptide yield can be expressed as the mass of peptide produced per unit volume (e.g., mg / liter or mg / L) or as the UV absorbance peak area of ​​the produced peptide in HPLC chromatography (e.g., mAu.sec). Cell density can be expressed as the visible light absorbance of the culture at a wavelength of 600 nm (OD600).

[0159] "OD" refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring the growth of a cell population over time, OD600 is preferred over UV spectroscopy. This is because cells are damaged under excessive UV light, and the 600 nm wavelength prevents this.

[0160] "OD660nm" or "OD 660nm This refers to the optical density at 660 nanometers (nm).

[0161] "Omega peptide," "omega toxin," "omega-ACTX-Hv1a," or "natural omega-ACTX-Hv1a" all refer to the ACTX peptide first isolated from the Australian blue mountain funnel-web spider known as Hadronyche versuta. The omega peptide is a positive allosteric regulator of the nicotinic acetylcholine receptor and also a voltage-gated Ca25 receptor in insects. 2+ Channel and voltage-dependent K +It may be a dual antagonist to the channel. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett. 2019 Jun;593(12):1336-1350, and Windley et al., Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors. Neuropharmacology. 2017 Dec;127:224-242 (their disclosures are incorporated herein by reference in their entirety).

[0162] A "single-letter code" refers to a peptide sequence that is listed by a single-letter code to distinguish various amino acids in the primary structure of a protein: alanine=A, arginine=R, asparagine=N, aspartic acid=D, asparagine or aspartic acid=B, cysteine=C, glutamic acid=E, glutamine=Q, glutamine or glutamic acid=Z, glycine=G, histidine=H, isoleucine=I, leucine=L, lysine=K, methionine=M, phenylalanine=F, proline=P, serine=S, threonine=T, tryptophan=W, tyrosine=Y, and valine=V.

[0163] "Operable" means the ability to be used, to do something, and / or to achieve some function or result. For example, in some embodiments, "operable" means the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to code for peptides, polypeptides, and / or proteins. For example, in some embodiments, a polynucleotide may be operable to code for a protein, meaning that the polynucleotide contains information to which it is embedded, along with the ability to produce a protein (for example, by transcribing mRNA which is subsequently translated into a protein).

[0164] "Operatively linked" refers to a juxtaposition of components described in this way that is in a relationship that enables them to function in the intended manner. For example, in some embodiments, operationally linked can refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operationally linked may mean that two adjacent DNA sequences are positioned together such that the transcriptional activation of one DNA sequence can act on the other DNA sequence. In yet another embodiment, the term "operatively linked" can refer to two or more peptides and / or polypeptides linked in such a way that they form a single polypeptide chain, or the term operationally linked can refer to two or more peptides linked in such a way that one peptide has some effect on the other. In yet another embodiment, operationally linked DNA sequences can refer to two adjacent DNA sequences being positioned together such that the transcriptional activation of one can act on the other.

[0165] An "ORF" or "open reading frame" refers to a sequence of RNA or DNA sequences between a translation initiation signal (e.g., AUG or ATG, respectively) and one or more known stop codons that code for one or more polypeptide sequences. In other words, an ORF describes a frame of reference from the perspective of a ribosome translating the RNA code, as long as the ribosome can continue reading (i.e., add amino acids to a nascent protein) because it has not yet encountered a stop codon. Thus, an "open reading frame" or "ORF" refers to the coded amino acid sequence between the translation start codon and stop codon of a coding sequence. Here, the terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence that designates the start and end of protein synthesis (mRNA translation), respectively.

[0166] In some embodiments, an ORF is a continuous sequence of codons beginning with a start codon (typically ATG in DNA and AUG in RNA) and ending with a stop codon (typically UAA, UAG, or UGA). In other embodiments, an ORF may be a sequence of RNA or DNA sequences between a translation initiation signal (e.g., AUG or ATG) and one or more known stop codons, the sequence of RNA or DNA sequences encoding one or more polypeptide sequences. In some other embodiments, an ORF may be a DNA sequence encoding a protein, beginning with an ATG start codon and ending with a TGA, TAA, or TAG stop codon. An ORF may also mean the translated protein encoded by the DNA. Generally, those skilled in the art distinguish the terms “open reading frame” and “ORF” from the term “coding sequence” based on the fact that the broadest definition of “open reading frame” simply intends a sequence of codons that does not contain a stop codon. Therefore, ORFs may contain introns, but coding sequences are distinguished by referring to those nucleotides (e.g., linked exons) that can be divided into codons that are actually translated into amino acids by the ribosome translation mechanism (i.e., coding sequences do not contain introns). However, as used herein, the terms “coding sequence,” “CDS,” “open reading frame,” and “ORF” are used interchangeably.

[0167] "Out-recombined" or "out-recombination" refers to the removal of a gene and / or polynucleotide sequence (e.g., an endogenous gene) adjacent to two site-specific recombination sites (e.g., the 5' and 3' nucleotide sequences of a target gene homologous to the homology arms of the target vector) during in vivo homologous recombination. See "knockout".

[0168] A "parasporal crystal toxin" refers to any peptide, polypeptide, or / or protein that is part of a parasporal body or parasporal crystal, which is a bipyramidal crystal containing one or more peptides, polypeptides, and / or proteins. When a parasporal body or parasporal crystal is ingested by an insect, the toxin-containing parasporal crystal dissolves in alkaline intestinal fluid and is then cleaved via a prototoxin midgut protease to produce an active peptide toxin (e.g., δ-endotoxin).

[0169] A "peptide expression cassette" or "expression cassette" means a DNA sequence consisting of all the DNA elements necessary to complete the transcription of an insecticidal protein in a biological expression system. The methods described herein include a transcription promoter, a DNA sequence encoding an α-junction signal sequence, a cleavage site, an insecticidal protein transgene, a stop codon, and a transcription terminator.

[0170] A "peptide expression vector" refers to an expression vector from a host organism that contains a heterologous peptide gene.

[0171] "Peptide-expressing yeast strain," "peptide-expressing strain," or "peptide-producing strain" refers to a yeast strain capable of producing heterologous peptides.

[0172] "Peptide-IA" refers to insecticides that are amino acids, peptides, polypeptides, and / or proteins.

[0173] A "peptide-transfer gene," "insecticide peptide-transfer gene," or "insecticide protein-transfer gene" refers to a DNA sequence that encodes a target peptide and can be translated in a biological expression system.

[0174] "Peptide yield" refers to the concentration of insecticidal peptides produced from the cells of a peptide-expressing yeast strain in the culture medium. This can be expressed as the mass of peptides produced per unit volume (e.g., mg / liter or mg / L) or as the UV absorbance peak area of ​​the peptides produced in an HPLC chromatograph (e.g., mAu.sec).

[0175] The "periphalic membrane" refers to the outer layer of the insect intestine, which protects the intestinal wall while enabling digestion and helping larger food particles move through the intestines.

[0176] "Pests" include, but are not limited to, insects, fungi, bacteria, nematodes, mites, and ticks.

[0177] "Pesticidally effective amount" refers to the amount of pesticide that can cause death to at least one pest or significantly reduce the growth, feeding, or normal physiological development of a pest. This amount will vary depending on factors such as the specific target pest being controlled, the specific environment, location, plant, crop, or agricultural land being treated, environmental conditions, and the method, rate, concentration, stability, and amount of application of the pesticide-effective polypeptide composition. Formulations may also vary with respect to climatic conditions, environmental considerations, and / or the frequency of application, and / or the severity of the pest infestation.

[0178] "Pharmacologically acceptable salt" is synonymous with agriculturally acceptable salt and, as used herein, refers to a compound modified by the preparation of its acid or base salt.

[0179] "Plant" means the entire plant, plant tissue, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, bulbils, embryos, and their offspring. Plant cells may be differentiated or undifferentiated (e.g., callus, suspension cells, plasma cells, leaf cells, root cells, phloem cells, and pollen).

[0180] A "plant transgene protein" refers to a heterologous protein that is expressed in a plant after the DNA or RNA encoding it has been delivered to one or more plant cells.

[0181] "Plant-cleavable linker" means a cleavable linker peptide, or a nucleotide encoding a cleavable linker peptide, that contains a plant protease recognition site and can be cleaved during the protein expression process in plant cells.

[0182] "Plant-integrated protective agents" or "PIPs" refer to insecticidal proteins produced by transgenic plants, and the genetic material required for the plant to produce those proteins.

[0183] A plasmid is a DNA segment that functions as a carrier for a target gene and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. A plasmid is a type of vector and can be a "cloning vector" (i.e., a simple plasmid used to clone a DNA fragment and / or to select a host population to carry the plasmid through some selection) or an "expression plasmid" (i.e., a plasmid used to produce large quantities of polynucleotides and / or polypeptides).

[0184] "Polar amino acids" are polar amino acids, including serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan, and tyrosine. Preferred polar amino acids are serine, threonine, cysteine, asparagine, and glutamine, with serine being the most preferred.

[0185] "Polynucleotide" refers to a polymeric form of nucleotides of any length (e.g., ribonucleotides, deoxyribonucleotides, or analogues) (e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides). As used herein, the term "polynucleotide" includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA, and also includes modified and unmodified forms of polynucleotides (modifications to and modifications of polynucleotides may include, for example, methylation, phosphorylation, and / or capping). In some embodiments, polynucleotides may be genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, or one of the aforementioned primers or amplified copies.

[0186] In yet another embodiment, polynucleotides may refer to a polymeric form of nucleotides capable of acting to encode the open reading frame of a gene.

[0187] In some embodiments, polynucleotides may refer to cDNA.

[0188] In some embodiments, polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The structure of a polynucleotide can be referred to by its 5' end, 3' end, or terminal, indicating the orientation of the polynucleotide. Adjacent nucleotides in a single strand of a polynucleotide are typically linked by phosphodiester bonds between their 3' and 5' carbons. However, bonds involving different nucleotides, such as methylene or phosphoramide bonds, can also be used. This means that each 5' and 3' carbon can be exposed at either end of the polynucleotide (these may be called the 5' end and the 3' end or terminal). The 5' and 3' ends may also be called the phosphoryl (PO4) end and the hydroxyl (OH) end, due to the chemical groups attached to each end. Furthermore, the term polynucleotide refers to both double-stranded and single-stranded molecules. Unless otherwise specified or requested, any embodiment of the construction or use of a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or expected to constitute the double-stranded form.

[0189] In some embodiments, polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs (including nucleotides with non-native bases, and nucleotides with modified native bases such as azapurines or deazapurines). If present, modifications to the nucleotide structure may be conferred before or after the assembly of the polynucleotide.

[0190] In some embodiments, polynucleotides may also be further modified after polymerization, for example, by conjugation with a labeling component. Furthermore, the sequence of nucleotides in the polynucleotide may be interrupted by non-nucleotide components. One or more ends of the polynucleotide may be protected or otherwise modified to prevent them from interacting with other polynucleotides in a particular way (e.g., forming covalent bonds).

[0191] In some embodiments, a polynucleotide may consist of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Uracil (U) may also be present as a natural substitution for thymine, for example, when the polynucleotide is RNA. Uracil may also be used in DNA. Therefore, the term “sequence” refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including both natural and non-natural bases.

[0192] The term "RNA molecule" or "ribonucleic acid molecule" refers to a polynucleotide having a ribose sugar instead of a deoxyribose sugar as one of its pyrimidine bases, and typically having uracil instead of thymine. The RNA molecules of the present invention are generally single-stranded, but may also be double-stranded. In the context of RNA molecules derived from an RNA sample, the RNA molecules may include single-stranded molecules transcribed from DNA in the cell nucleus, mitochondria, or chloroplasts, which have a linear sequence of nucleotide bases complementary to the DNA strand from which they are transcribed.

[0193] In some embodiments, the polynucleotide may further comprise one or more heterogeneous regulatory elements. For example, in some embodiments, the regulatory elements may be one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA transport elements, internal ribosome entry sites (IRESs), poly-U sequences, or combinations thereof.

[0194] Post-transcriptional gene silencing, or PTGS, refers to a cellular process within living cells that suppresses gene expression.

[0195] A "post-transcriptional regulatory element" is a DNA segment and / or mechanism that affects mRNA after it has been transcribed. Post-transcriptional mechanisms include splicing events, capping, poly(A) tail addition, and other mechanisms known to those skilled in the art.

[0196] "Promoter" refers to the region of DNA to which RNA polymerase binds to initiate transcription of a gene.

[0197] "Protein" has the same meaning as "peptide" and / or "polypeptide" in this document.

[0198] "Ratio" refers to the quantitative relationship between two quantities, indicating how many times one value is contained in or included in the other value.

[0199] "Reading frame" refers to one of the six (three in each direction) possible reading frames of a double-stranded DNA molecule. The reading frame used determines which codons are used to encode amino acids within the coding sequence of the DNA molecule. In some embodiments, a reading frame is a way of dividing the nucleotide sequence in a polynucleotide and / or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non-overlapping triplets.

[0200] "Recombinant DNA" or "rDNA" refers to DNA consisting of two or more different DNA segments.

[0201] "Recombinant vector" means a DNA plasmid vector into which foreign DNA has been inserted.

[0202] "Regulatory element" refers to a genetic element that controls some aspects of the expression and / or processing of a nucleic acid sequence. For example, in some embodiments, regulatory elements can be found at the transcriptional and post-transcriptional levels. A regulatory element can be a cis-regulatory element (CRE) or a trans-regulatory element (TRE). In some embodiments, the regulatory element can be one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA transport elements, internal ribosome entry sites (IRES), poly-U sequences, and / or other elements that affect gene expression (e.g., increase or decrease expression and / or cause constitutive expression in a tissue-specific, time-dependent manner).

[0203] "Restriction enzyme" or "restriction endonuclease" refers to an enzyme that cuts DNA at a specified restriction site. For example, a restriction enzyme can cut a plasmid at an EcoRI, SacII, or BstXI restriction site to linearize the plasmid and ligate the DNA of interest.

[0204] "Restriction site" refers to a position on DNA that contains a sequence of 4 to 8 nucleotides that is recognized by a specific restriction enzyme.

[0205] "Salannin" refers to a compound isolated from Azadirachta indica that has insecticidal activity. In some embodiments, salannin has a molecular formula of C 34 H 44 O9 and a molecular weight of 596.7 g / mol.

[0206] The term "sea anemone" refers to a group of marine animals belonging to the order Anemoniformes. Sea anemones are named after the terrestrial flowering plant Anemone, due to the diverse appearances of many species. For example, in one embodiment, a sea anemone is one of the following species: Actinia equine, Anemonia erythraea, Anemonia sulcata, Anemonia viridis, Anthopleura elegantissima, Anthopleura fuscoviridis, Anthopleura xanthogrammica, Bunodosoma caissarum, Bunodosoma cangicum, Bunodosoma granulifera, Heteractis crispa, Parasicyonis actinostoloides, Radianthus paumotensis, or Stoichactis helianthus.

[0207] A "selection gene" refers to a gene that confers an advantage to genetically modified organisms, allowing them to reproduce under selective pressure.

[0208] A "serovar" or "serotype" refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, serovars are a variety of microorganisms that are antigenically and serologically distinct.

[0209] "sp." refers to a species.

[0210] "ssp." or "subsp." refers to a subspecies.

[0211] "Subcloning" or "subcloned" refers to the process of transferring DNA from one vector to another, usually a more favorable vector. For example, a polynucleotide encoding a mutant or peptide can be subcloned into the pKlac1 plasmid, and then yeast colonies transformed with the pKLAC1 plasmid can be selected.

[0212] "SSI" is a context-dependent acronym. In some contexts, it can refer to "site-directed recombination," used to refer to a sequence that allows homologous recombination in vivo to occur at a specific site within the genome of a host organism. Thus, in some embodiments, the term "site-directed recombination" refers to the process of directing a transgene to a target site within the genome of a host organism, allowing the desired gene to be integrated into a pre-selected genomic location in the host organism. However, in other contexts, SSI can refer to "surface spraying indoors," a technique that applies a variable amount of insecticide to surfaces inhabited by vectors, such as walls, windows, floors, and ceilings.

[0213] "STA," or "translation-stabilizing protein," or "stabilizing domain," or "stabilizing protein" (as used interchangeably herein) means a peptide or protein having sufficient tertiary structure to accumulate in cells without being targeted by cellular processes of proteolysis. The protein may be 5 to 50 amino acids long. The translation-stabilizing protein is encoded by a DNA sequence of a protein that is operably ligated to a sequence encoding the insecticidal protein or CRIP of the ORF. The operably ligated STA may be either upstream or downstream of CRIP and may have any intervening sequence between the two sequences (STA and CRIP), as long as the intervening sequence does not result in a frameshift of either DNA sequence. The translation-stabilizing protein may also have activity to increase the delivery of CRIP across the intestinal wall to the insect hemolymph.

[0214] "sta" refers to a nucleotide that codes for translation stabilization proteins.

[0215] A "structural motif" refers to the three-dimensional arrangement of a peptide and / or polypeptide, and / or the arrangement of operable linked polypeptide segments. For example, a polypeptide having an ERSP motif, an STA motif, a linker motif, and a CRIP polypeptide motif has the overall "structural motif" of ERSP-STA-L-CRIP. See also "CRIP construct".

[0216] "Ta1b," "U1-agatoxin-Ta1b," "Ta1bWT," or "wild-type U1-agatoxin-Ta1b" refers to polypeptides isolated from the hobo spider *Eratigena agrestis*. An example of U1-agatoxin-Ta1b is the polypeptide with the amino acid sequence of SEQ ID NO: 1 (NCBI accession number O46167.1).

[0217] "Ta1b variant polynucleotide" or "U1-agatoxin-Ta1b variant polynucleotide" refers to a polynucleotide or group of polynucleotides capable of expressing and / or encoding an insecticidal protein containing one or more TVPs. The term "U1-agatoxin-Ta1b variant polynucleotide" is used to describe a U1-agatoxin-Ta1b variant polynucleotide sequence contained in a TVP expression ORF, when it is contained in a vector, and / or when it describes a polynucleotide encoding an insecticidal protein, it is described as "tvp" and / or "Tvp".

[0218] "Toxin" refers to venomous fluids and / or poisons, in particular proteins or conjugate proteins produced by certain animals, higher plants, and pathogenic bacteria. Generally, the term "toxin" refers to molecules and peptides found in natural products, such as scorpions, spiders, snakes, and poisonous mushrooms, while the term "poison" refers to man-made products and / or artificial products, such as synthetic chemical pesticides. However, as used herein, the terms "toxin" and "toxic substance" are used synonymously.

[0219] Both “transfection” and “transformation” refer to the process of introducing exogenous and / or heterologous DNA or RNA (e.g., a vector containing polynucleotides encoding CRIP) into a host organism (e.g., a prokaryote or a eukaryote). Generally, those skilled in the art may reserve the term “transformation” to describe the process of introducing exogenous and / or heterologous DNA or RNA into bacterial cells, and the term “transfection” to describe the process of introducing exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms “transformation” and “transfection” are used synonymously regardless of whether the process describes the introduction of exogenous and / or heterologous DNA or RNA into a prokaryote (e.g., a bacterium) or a eukaryote (e.g., yeast, a plant, or an animal).

[0220] A "transgene" refers to a heterologous DNA sequence that codes for a protein that is transformed into a plant.

[0221] A "transgenic host cell" refers to a cell that has been genetically transformed and selected for its transgenic state via additional selection genes.

[0222] A "transgenic plant" refers to a plant derived from a single cell that has been transformed with foreign DNA so that all of the plant's cells contain that transgene.

[0223] The "transient expression system" refers to an Agrobacterium tumefaciens-based system that delivers DNA encoding a disarmed plant virus to the plant cells in which it is to be expressed. The plant virus has been engineered to express the protein of interest at high levels, up to 40% of the total soluble protein (TSP).

[0224] The "triple expression cassette" refers to three CRIP expression cassettes contained on the same vector.

[0225] "TRBO" refers to a transient plant expression system using a tobacco mosaic virus from which the viral coat protein gene has been removed.

[0226] "TSP" or "total soluble protein" refers to the total amount of protein extracted from a plant tissue sample and solubilized in the extraction buffer.

[0227] "TVP" or "U1-agatoxin-Ta1b variant polypeptide (TVP)" or "Ta1b variant polypeptide (TVP)" refers to a variant or variant of the wild-type U1-agatoxin-Ta1b polypeptide sequence and / or the polynucleotide sequence encoding the wild-type U1-agatoxin-Ta1b polypeptide, which has been modified to produce a polypeptide and / or polynucleotide sequence that does not exist in nature. An exemplary wild-type U1-agatoxin-Ta1b polypeptide sequence having the amino acid sequence of SEQ ID NO: 1 is provided herein. An exemplary wild-type U1-agatoxin-Ta1b precursor polypeptide sequence is provided herein, having the amino acid sequence of SEQ ID NO: 48 (NCBI accession number O46167.1) and including the signal sequence "MKLQLMICLVLLPCFFC" (SEQ ID NO: 59). In some embodiments, TVP may have an amino acid sequence of any of the amino acid sequences listed in Table 1. Thus, the term "TVP" refers to a peptide having one or more mutations compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, TVP may have an amino acid sequence according to formula (I). EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 Equation (I)

[0228] In the formula, the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0229] In some embodiments, TVP may have an amino acid sequence according to formula (II), or a pharmaceutically acceptable salt thereof. EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG Formula (II)

[0230] In the formula, the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A.

[0231] "U-ACTX-Hv1a," "hybrid peptide," "hybrid toxin," "hybrid-ACTX-Hv1a," "natural hybrid ACTX-Hv1a," "U peptide," "U toxin," "natural U," or "natural U-ACTX-Hv1a" all refer to the ACTX peptide, which was discovered in a spider known as the Australian blue mountain funnel spider, Hadronyche versuta. U-ACTX-Hv1a is a positive allosteric regulator of the nicotinic acetylcholine receptor and also affects insect voltage-gated Ca 2+ Channel and voltage-dependent K +It may be a dual antagonist to the channel. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett. 2019 Jun;593(12):1336-1350, and Windley et al., Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors. Neuropharmacology. 2017 Dec;127:224-242 (their disclosures are incorporated herein by reference in their entirety). An exemplary U-ACTX-Hv1a peptide is provided in SEQ ID NO: 60.

[0232] "U+2 peptide," "U+2 protein," "U+2 toxin," "U+2," "U+2-ACTX-Hv1a," or "Spear" all refer to U-ACTX-Hv1a having an additional dipeptide operably linked to the native peptide. The additional dipeptide operably linked to the U peptide is indicated by "+2" or "plus 2" and can be selected from several peptides, any of which may result in a "U+2 peptide" having the unique properties discussed herein. In some preferred embodiments, the dipeptide is "GS," and an exemplary U+2-ACTX-Hv1a peptide is shown in SEQ ID NO: 61.

[0233] "UBI" refers to ubiquitin. For example, in some embodiments, UBI may refer to a ubiquitin monomer isolated from Zea mays.

[0234] "var." refers to a variety or cultivar. The term "var." is used to indicate a taxonomic category ranked at the species level and / or subspecies (if any) below. In some embodiments, the term "var." refers to a member that differs from other members of the same subspecies or species in minor but persistent or genetic characteristics.

[0235] A “variant,” “variant sequence,” or “variant peptide” refers to an amino acid sequence having one or more conserved amino acid substitutions or modifications. Conservative amino acid substitutions in a “variant” do not substantially reduce the activity of the variant in relation to the non-variable form of the variant. For example, in some embodiments, a “variant” has one or more conserved amino acid substitutions compared to a peptide having the disclosed sequence and / or claimed sequence as indicated by the SEQ ID NO:

[0236] A "vector" refers to a DNA segment that accepts a target foreign gene (e.g., a crip). The target gene is known as an "insertion" or "transgene."

[0237] "Vip," or "VIP," or "plant insecticidal protein" refers to proteins discovered by screening the supernatant of plant-grown Bt strains for possible insecticidal activity. Vips are little to no similar to Cry proteins. In particular, the use and preferred use in this document refers to the VIP3 or Vip3 protein, which has lepidopteran activity. Vip is thought to have a similar mode of action to the Bt cry peptide.

[0238] "Vitrification" refers to the process of converting a material into a glassy amorphous material. A glassy amorphous solid does not necessarily have to contain any crystalline structure. Solidification of the glassy solid occurs at the glass transition temperature (Tg).

[0239] "Wild type" or "WT" refers to the phenotype and / or genotype (i.e., appearance or sequence) of an organism, polynucleotide sequence, and / or polypeptide sequence as found and / or observed in its naturally occurring state or conditions.

[0240] "Yeast expression vector," "expression vector," or "vector" means a plasmid that can introduce heterologous genes and / or expression cassettes into yeast cells that are transcribed and translated.

[0241] "Yield" refers to the production of peptides, and an increase in yield may mean an increase in the amount produced, an increase in the rate of production, an increase in the mean or median yield, and an increase in the frequency of higher yields. The term "yield," as in "plant yield," refers to the quality and / or quantity of biomass produced by a plant when used in relation to the growth and / or production of a plant crop.

[0242] Throughout this specification, unless otherwise specified or unless the context requires otherwise, a single step, composition of a substance, a group of steps, or a group of compositions of a substance shall be deemed to encompass one and more (i.e., one or more) of those steps, compositions of a substance, a group of steps, or a group of compositions of a substance.

[0243] This disclosure is carried out without excessive experimentation using conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solid-phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid-phase peptide synthesis, immunology, cell culture, and formulation, unless otherwise indicated. Such procedures are described in, for example, all volumes of Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), Volumes I, II, and III; all texts of DNA Cloning: A Practical Approach, Vols. I and II (DNGlover, ed., 1985), IRL Press, Oxford; all texts of Oligonucleotide Synthesis: A Practical Approach (MJ Gait, ed., 1984), IRL Press, Oxford; and in particular, the papers by Gait, pp. 1-22, Atkinson et al, pp. 35-81, Sproat et al, pp. 83-115, and Wu et al, pp. 135-151, and 4. Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press. Press, Oxford, All texts, Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, All texts, Perbal, B., A Practical Guide to Molecular Cloning (1984), Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), All series, JFRamalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany), Sakakibara, D., Teichman, J., Lien, E. L. and Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73, 336 - 342, Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149 - 2154, Barany, G. and Merrifield, R. B. (1979) in “The Peptides” (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1 - 284, Academic Press, New York. 12. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben - Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th ed., Parts 1 and 2, Thieme, Stuttgart, Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer - Verlag, Heidelberg, Bodanszky, M.& Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer - Verlag, Heidelberg, Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449 - 474, Handbook of Experimental Immunology, Vols. I - IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications), and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed.(As described in ,2000) (each of these references is incorporated herein by reference in its entirety).

[0244] Throughout this specification, unless otherwise specified in the context, variations such as “comprise,” “comprises,” or “comprising” shall mean the inclusion of the steps, elements, or integers described, or a group of steps, elements, or integers, but not the exclusion of any other steps, elements, or integers, or a group of elements or integers.

[0245] All patent applications, patents, and publications referenced herein are incorporated by reference in whole to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by whole. Furthermore, all patent applications, patents, and publications cited herein are incorporated by whole by whole, except for any definitions, subject matter disclaimers, or denials, and unless the incorporated material conflicts with the disclosures herein (in which case the language of these disclosures shall prevail).

[0246] Cysteine-rich insecticidal protein (CRIP) The present invention provides combinations comprising (1) one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticide proteins or pharmaceutically acceptable salts thereof, or a combination thereof, and (2) one or more insecticides (IA). Several types of CRIPs are intended and taught herein. The CRIPs of the present invention can be used in combination with the insecticides (IA) of the present invention, which are described in detail below. All CRIPs intended below, which are suitable for the combinations of the present invention, contain CRIP-insecticide proteins.

[0247] Spider peptides and toxins In some embodiments, CRIP may be a spider toxin peptide or protein isolated from one of the following: Phoneutria nigriventer, Allagelena opulenta, Cupiennius salei, Plectreurys tristis, Coremiocnemis valida, Haplopelma huwenum, Agelena orientalis, Allagelena opulenta, Segestria florentina, Apomastus schlingeri, Phoneutria keyserlingi, Macrothele gigas, Macrothele raveni, Missulena bradleyi, Pireneitega luctuosa, Phoneutria reidyi, Illawara wisharti, Eucratoscelus constrictus, Ageenopsis aperta, Hololena curta, Oxyopes lineatus, Brachypelma albiceps, or Brachypelma smithi.

[0248] In some embodiments, CRIP may be isolated from Hadronyche versuta or blue mountain funnel-web spider, Hadronyche venenata, Atrax robustus, Atrax formidabilis, or Atrax infensus.

[0249] In some embodiments, CRIP may be any of the following spider peptides, polypeptides, and / or toxins: U+2-ACTX-Hv1a; Γ-CNTX-Pn1a; U13-ctenitoxin-Pn1a, U13-ctenitoxin-Pn1b, U13-ctenitoxin-Pn1c, U1-agatoxin-Aop1a, U1-ctenitoxin-Cs1a, U1-nemethoxine-Csp1a, U1-nemethoxine-Csp1b, U1-nemethoxine-Csp1c, U1-plectoxin-Pt1a, U1-plectoxin -Pt1b, U1-plectoxin-Pt1c, U1-plectoxin-Pt1d, U1-plectoxin-Pt1f, U1-seraphotoxin-Cv1a, U1-seraphotoxin-Hh1a_1, U1-seraphotoxin-Hh1a_2, U1-seraphotoxin-Hh1a_3, U1-seraphotoxin-Hh1b, U1-seraphotoxin-Hh1c_1, U1-seraphotoxin-Hh1c_2, U1-seraphotoxin-Hh1d, U1-seraphotoxin-Hh1e, U1-seraphotoxin-Hh1f_1, U1-seraphotoxin-Hh1f_2, U1-seraphotoxin U2-Segestritoxin-Hh1f_3, U2-Segestritoxin-Hh1f_4, U2-Segestritoxin-Hh1g, U2-Agatoxin-Ao1a, U2-Agatoxin-Aop1a, U2-Ctenitoxin-Cs1a, U2-Ctenitoxin-Pn1a, U2-Siltautoxin-As1a, U2-Segestritoxin-Sf1a, U2-Segestritoxin-Sf1b, U2-Segestritoxin-Sf1c, U2-Segestritoxin-Sf1d, U2-Segestritoxin-Sf1e, U2-Segestritoxin-Sf1f, U2-Segestritoxin-S f1g, U2-segestritoxin-Sf1h, U2-seraphotoxin-Hh1a, U3-siltautoxin-As1a, U3-plectoxin-Pt1a, U5-ctenitoxin-Pn1a, U7-ctenitoxin-Pk1a, β-hexatoxin-Mg1a, β-hexatoxin-Mr1a, Γ-ctenitoxin-Pn1a, δ-actinopositoxin-Mb1a, δ-amaurobitoxin-Pl1a, δ-amaurobitoxin-Pl1b, δ-amaurobitoxin-Pl1c, δ-amaurobitoxin-Pl1d, δ-ctenitoxin-Asp2e,δ-Ctenitoxin-Pn1a_1, δ-Ctenitoxin-Pn1a_2, δ-Ctenitoxin-Pn1b, δ-Ctenitoxin-Pn2a, δ-Ctenitoxin-Pn2b, δ-Ctenitoxin-Pn2c, δ-Ctenitoxin-Pr2d, δ-Hexatoxin-Ar1a, δ-Hexatoxin-Hv1a, δ-Hexatoxin-Hv1b, δ-Hexatoxin-Iw1a, δ-Hexatoxin-Mg1a, δ-Hexatoxin-Mg1b, κ-Hexatoxin-Hf1a, κ-Hexatoxin-Hv1a, κ-Hexatoxin-Hv1b, κ-Hexatoxin-Hv1c_1, κ-Hexatoxin-Hv1c_2, κ-Hexatoxin-Hv1c_3, κ-Hexatoxin-Hv1c_4, κ-Hexatoxin-Hv1d, κ-Hexatoxin-Hv1e, κ-Seraphotoxin-Ec2a, κ-Seraphotoxin-E c2b, μ-agatoxin-Aa1a, μ-agatoxin-Aa1b, μ-agatoxin-Aa1c, μ-agatoxin-Aa1d, μ-agatoxin-Aa1e, μ-agatoxin-Aa1f, μ-agatoxin-Hc1a, μ-agatoxin-Hc1b, μ-agatoxin-Hc1c, μ-hexatoxin-Mg1a, μ-hexatoxin-Mg1b, μ-hexatoxin-Mg1c, μ-hexatoxin n-Mg2a, μ-seraphotoxin-Hh1a, ω-actinopositoxin-Mb1a, ω-agatoxin-Aa4a, ω-agatoxin-Aa4b, ω-agatoxin-Aa4c, ω-hexatoxin-Ar1a_1, ω-hexatoxin-Ar1a_3, ω-hexatoxin-Ar1b_1, ω-hexatoxin-Ar1d_1, ω-hexatoxin-Ar1d_4, ω-hexatoxin-Ar1e_1 ω-hexatoxin-Ar1f, ω-hexatoxin-Ar1g_1, ω-hexatoxin-Ar1h, ω-hexatoxin-Ar2a, ω-hexatoxin-Ar2b, ω-hexatoxin-Ar2c, ω-hexatoxin-Ar2d, ω-hexatoxin-Ar2e_1, ω-hexatoxin-Ar2e_2, ω-atlachotoxin-Asp2a, ω-hexatoxin-Asp2b, ω-hexatoxin-Hf1a, ω-hexatoxin-Hi1a_1, ω-hexatoxin-Hi1a_2, ω-hexatoxin-Hi1a_3, ω-hexatoxin-Hi1b_1,ω-hexatoxin-Hi1b_10, ω-hexatoxin-Hi1b_2, ω-hexatoxin-Hi1b_5, ω-hexatoxin-Hi1b_8, ω-hexatoxin-Hi1c_1, ω-hexatoxin-Hi1c_2, ω-hexatoxin-Hv1a, ω-hexatoxin-Hv1b, ω-hexatoxin-Hv1c, ω-hexatoxin-Hv1d, ω-hexatoxin-Hv1e, ω-hexatoxin-Hv1f, ω-hexatoxin-Hv1g_1, ω-hexatoxin-Hv1g_5, ω-hexatoxin-Hv1 g_6ω-hexatoxin-Hv2a, ω-hexatoxin-Hv2b_1, ω-hexatoxin-Hv2b_2, ω-hexatoxin-Hv2b_3, ω-hexatoxin-Hv2b_4, ω-hexatoxin-Hv2b_5, ω-hexatoxin-Hv2b_6, ω-hexatoxin-Hv2b_7, ω-hexatoxin-Hv2c, ω-hexatoxin-Hv2d_1, ω-hexatoxin-Hv2d_2, ω-hexatoxin-Hv2d_3, ω-hexatoxin-Hv2e, ω-hexatoxin-Hv2f, ω-hexato Hv2g, ω-hexatoxin-Hv2h_1, ω-hexatoxin-Hv2h_2, ω-hexatoxin-Hv2i, ω-hexatoxin-Hv2j_1, ω-hexatoxin-Hv2j_2, ω-hexatoxin-Hv2k, ω-hexatoxin-Hv2l, ω-hexatoxin-Hv2m_1, ω-hexatoxin-Hv2m_2, ω-hexatoxin-Hv2m_3, ω-hexatoxin-Hv2n, ω-hexatoxin-Hv2o, ω-hexatoxin-Hvn1a, ω-hexatoxin-Hvn1b_1, ω- Hexatoxin-Hvn1b_2, ω-hexatoxin-Hvn1b_3, ω-hexatoxin-Hvn1b_4, ω-hexatoxin-Hvn1b_6, ω-hexatoxin-Iw2a, ω-oxotoxin-Ol1b, ω-plectoxin-Pt1a, ω-seraphotoxin-Asp1a, ω-seraphotoxin-Asp1f, ω-seraphotoxin-Asp1g, ω-seraphotoxin-Ba1a, ω-seraphotoxin-Ba1b, ω-seraphotoxin-Bs1a, ω-seraphotoxin-Bs2a, or ω-seraphotoxin-Hh2a.

[0250] In some embodiments, CRIP may be a spider toxin peptide or protein having the amino acid sequence shown in any one of SEQ ID NOs: 192-278 and 281-370.

[0251] In some cases, the polynucleotide encoding CRIP can encode a CRIP having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 192-278 and 281-370.

[0252] ACTX peptide In some embodiments, CRIP may be an ACTX peptide.

[0253] In some embodiments, CRIP may be one or more of the following ACTX peptides: U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, rκ-ACTX-Hv1c, ω-ACTX-Hv1a, and / or ω-ACTX-Hv1a+2.

[0254] Exemplary ACTX peptides include: U-ACTX-Hv1a having the amino acid sequence "QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA" (SEQ ID NO: 60), U+2-ACTX-Hv1a having the amino acid sequence "GSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA" (SEQ ID NO: 61), and the amino acid sequence "SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD" (SEQ ID NO: 60). Omega-ACTX-Hv1a having number 62), "ω+2-ACTX-Hv1a+2" (or "Omega+2-ACTX-Hv1a") having the amino acid sequence "GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD" (sequence number 63), and kappa+2-ACTX-Hv1a (or κ+2-ACTX-Hv1a) having the amino acid sequence "GSAICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP" (sequence number 64).

[0255] In some embodiments, CRIP may be "kappa-ACTX-Hv1a" (or κ+2-ACTX-Hv1a) having the amino acid sequence "AICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP" (SEQ ID NO: 594).

[0256] In some embodiments, the ACTX peptide may contain an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with respect to SEQ ID NOs. 60-64, 192-370, and 594.

[0257] In some cases, the polynucleotide encoding the ACTX peptide can encode an ACTX peptide having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 60-64 and 594.

[0258] CNTX-Pn1a peptide In some preferred embodiments, CRIP may be a Γ-CNTX-Pn1a or γ-CNTX-Pn1a toxin. The Γ-CNTX-Pn1a peptide is an insecticidal neurotoxin derived from the Brazilian armed spider Phoneutria nigriventer. Γ-CNTX-Pn1a targets the N-methyl-D-aspartate (NMDA) subtype ion channel glutamate receptor (GRIN) and sodium channels. An exemplary wild-type full-length Γ-CNTX-Pn1a peptide has the following amino acid sequence: MKVAIVFLSLLVLAFASESIEENREEFPVEESARCADINGACKSDCDCCGDSVTCDCYWSDSCKCRESNFKIGMAIRKKFC (SEQ ID NO: 689) (NCBI accession number P59367). A recombinant mature Γ-CNTX-Pn1a peptide having the amino acid sequence "GSCADINGACKSDCDCCGDSVTCDCYWSDSCKCRESNFKIGMAIRKKFC" (SEQ ID NO: 65) is provided.

[0259] In some cases, the Γ-CNTX-Pn1a peptide may contain an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to Sequence ID No. 65.

[0260] In some embodiments, the polynucleotide encoding the Γ-CNTX-Pn1a peptide is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least It is possible to encode a Γ-CNTX-Pn1a peptide having an amino acid sequence that is 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical.

[0261] Wild-type U1-agatoxin and TVP The hobo spider (Eratigena agrestis, formerly Tegenaria agrestis) is a venomous spider belonging to the family Agelenidae, specifically a funnel-web spider. See Ingale A, Antigenic epitopes prediction and MHC binder of a paralytic insecticidal toxin (ITX-1) of Tegenaria agrestis (hobo spider). 4 August 2010 Volume 2010:2 pp 97-103. The venom of the hobo spider is considered to have insecticidal activity. Johnson et al.,Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system.Arch Insect Biochem Physiol.1998;38(1):19-31, Klint et al.,Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E.coli.PLoS Please refer to One.2013;8(5):e63865.

[0262] Hobo spiders, along with several other spiders in the family Agelenidae, produce a venom containing agatoxin, which exhibits insecticidal activity. Agatoxin is a group of chemically diverse toxins that can induce various insecticidal effects depending on the target species. For example, agatoxin causes delayed spastic paralysis in Coleoptera, Lepidopteran, and Diptera, increases the rate of neuronal firing in the central nervous system (CNS) of houseflies (Musca domestica), and is lethal to other insects (e.g., blowflies, Lucilia cuprina). Thus, agatoxin is involved in the targeting of the CNS. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators. Structure 23:1283-1292, and Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19-31 (1998).

[0263] Two types of agatoxins are U1-agatoxin-Ta1a and U1-agatoxin-Ta1b, both members of the Helical Arthropod Neuropeptide (HAND) toxin family. In addition to spiders, these toxins are also found in the venom of centipedes. Agatoxins are evolutionary derivatives of the ancient molting animal hormone family, namely the ion transport peptide / crustacean blood glucose-raising hormone (ITP / CHH) family. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators. Structure 23:1283-1292, and Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19-31 (1998).

[0264] The hobo spider-derived U1-agatoxin-Ta1b toxin has the full-length amino acid sequence "MKLQLMICLVLLPCFFCEPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQKG (SEQ ID NO: 48)" and contains a signal peptide from amino acid positions 1-17 and a mature toxin from positions 18-68. (Ibid.) The protein contains four densely packed α-helices without a β-chain, and the molecular weight of the mature toxin is 5700.39 daltons (Da). (Ibid.)

[0265] An exemplary mature wild-type U1-agatoxin-Ta1b polypeptide from Eratigena agrestis is provided, having the following amino acid sequence: "EPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQKG" (SEQ ID NO: 1).

[0266] During protein processing, the mature wild-type U1-agatoxin-Ta1b toxin undergoes a cleavage event of the C-terminal glycine, resulting in the following amino acid sequence: EPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQK (SEQ ID NO: 60). Subsequent post-translational events yield the mature wild-type U1-agatoxin-Ta1b toxin with C-terminal amidation.

[0267] U1-agatoxin-Ta1b variant polypeptides (TVPs) are variants or variants that differ in some way from wild-type U1-agatoxin-Ta1b (SEQ ID NO: 1). For example, in some embodiments, this difference may be an amino acid substitution, deletion, or addition, or modification, to the polynucleotide encoding wild-type U1-agatoxin-Ta1b. The result of this variation is a naturally occurring polypeptide and / or the polynucleotide sequence encoding it, which has enhanced insecticidal activity against one or more insect species compared to wild-type U1-agatoxin-Ta1b.

[0268] In some embodiments, the TVP may have the amino acid sequence of SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654, as shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0269] In some embodiments, polynucleotide sequences having sequences 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654 can be configured to encode TVP. For example, in some embodiments, the polynucleotides shown in Table 2 can be configured to encode TVP. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0270] Exemplary TVP Exemplary descriptions of TVPs and polynucleotides capable of encoding TVPs are provided in International Application No. PCT / US21 / 28254 (the disclosure thereof is incorporated herein by reference in its entirety).

[0271] In some embodiments, the TVP contains one or more mutations compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. For example, in some embodiments, the TVP may have a first, second, or third mutation compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1.

[0272] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, and at least 9 A TVP containing an amino acid sequence that is 9.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, or a pharmaceutically acceptable salt thereof: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide is the wild-type U1-agatoxin-Ta1b shown in SEQ ID NO: 1) The sequence contains at least one amino acid substitution compared to the given sequence, wherein X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0273] In some implementations, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, and at least 99.8% identical. 1. A TVP containing an amino acid sequence that is at least 99.9% identical or 100% identical, or a pharmaceutically acceptable salt thereof: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, X1 X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; and TVP has one amino acid substitution in X1, X2, X3, X4, or X5.

[0274] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, and less TVPs containing amino acid sequences that are 99.9% or 100% identical, or pharmaceutically acceptable salts thereof, may be: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or (N is X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is glycine).

[0275] In some implementations, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, and less TVPs containing amino acid sequences that are at least 99.9% identical or 100% identical, or pharmaceutically acceptable salts thereof, may be: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or (where is N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is absent).

[0276] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least TVPs containing amino acid sequences that are 99.9% identical or 100% identical, or pharmaceutically acceptable salts thereof, may exist: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N) (Yes, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; TVP has one amino acid substitution in X1, X2, X3, X4, or X5; and X6 and X7 are absent).

[0277] In some implementations, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical. A TVP containing the amino acid sequence, or a pharmaceutically acceptable salt thereof, may be: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, V, M, (The amino acid sequence is I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP contains the amino acid sequence shown in any one of SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654).

[0278] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, and at least 99.9% identical. Alternatively, it may be a TVP containing an amino acid sequence that is 100% identical, or a pharmaceutically acceptable salt thereof: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, or N) (where X3 is A, G, N, L, D, V, M, I, C, E, T, or S, X4 is T or P, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP is encoded by a polynucleotide sequence shown in any one of sequence numbers 17-30, 54-58, or 655-688).

[0279] In some implementations, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, and at least 99.8% identical. A TVP may contain an amino acid sequence that is at least 99.9% identical or 100% identical: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q) X3 is N, A, G, N, L, D, V, M, I, C, E, T, or S, X4 is T or P, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP further comprises two or more homopolymers or heteropolymers of TVP, where the amino acid sequences of each TVP are the same or different).

[0280] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, and at least 99.9% identical. A TVP may contain an amino acid sequence that is % identical or 100% identical: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, (V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, where the amino acid sequences of each TVP may be the same or different).

[0281] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical. A TVP may contain the following amino acid sequence: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S) (where X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, the amino acid sequences of each TVP may be the same or different, and the linker is cleavable in the intestine or hemolymph of an insect).

[0282] In some embodiments, the linker has the amino acid sequence shown in any one of SEQ ID NOs: 61 to 70.

[0283] In some cases, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, and at least 99 A TVP containing an amino acid sequence that is 0.8% identical, at least 99.9% identical, or 100% identical, or a pharmaceutically acceptable salt thereof: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 (wherein the polypeptide, at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1) (TVP is glycosylated if it includes the following: X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; and Z1 is T or S.)

[0284] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, and less than 50% identical. It may be a TVP containing an amino acid sequence that is at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical.

[0285] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, and at least 95% identical. 1. A TVP containing an amino acid sequence that is at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, or a pharmaceutically acceptable salt thereof: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG (wherein the formula, the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A).

[0286] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, and less TVPs containing amino acid sequences that are at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, or pharmaceutically acceptable salts thereof: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG (wherein the formula, the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, Z1 is T or A, and if Z1 is T, the TVP is glycosylated).

[0287] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, and at least 96% identical. TVPs containing amino acid sequences that are % identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, or pharmaceutically acceptable salts thereof: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG (wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, Z1 is T or A, X1 is Q, and Z1 is A).

[0288] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, and at least TVPs containing amino acid sequences that are 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, or pharmaceutically acceptable salts thereof.

[0289] Until some TVPs are fully realized, they are at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, and at least 92% identical. May contain amino acid sequences that are % identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical: "EPDEICRAQMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYAACHEAQKG" (SEQ ID NO: 51).

[0290] In some preferred embodiments, the TVP may be TVP-R9Q / T43A (SEQ ID NO: 51).

[0291] In various embodiments, polynucleotides encoding TVP can be used to transform plant cells, yeast cells, or bacterial cells. In some embodiments, the insecticidal TVP transgenic protein can be formulated into a composition that can be sprayed onto the surface of a plant or a part thereof, or otherwise applied, in any manner known to those skilled in the art. Thus, DNA constructs capable of operating to encode one or more TVPs under appropriate conditions in host cells, e.g., plant cells, are provided herein. A method for controlling insect pest infection of plant cells by parasitic insects comprises administering or introducing polynucleotides encoding TVP as described herein into a plant, plant tissue, or plant cell by recombinant technology, and growing the recombinantly modified plant, plant tissue, or plant cell in the field exposed to the pest. Alternatively, TVP can be formulated into a spray composition comprising TVP and excipients, and can be applied directly to susceptible plants by direct application to produce a harmful effect upon ingestion of TVP by infectious insects.

[0292] Scorpion peptides and toxins In some embodiments, CRIP may be any of the following scorpion peptides, polypeptides, and / or toxins: imperatoxin-A (IpTxa), potassium channel toxin α-KTx10.2 (cobatoxin-2), potassium channel toxin α-KTx11.1 (parabutoxin-1), potassium channel toxin α-KTx11.2 (parabutoxin-2), potassium channel toxin α-KTx11.3 (parabutoxin-10), potassium channel toxin α-KTx12.1 (butantoxin), potassium channel toxin α-KTx12 .2 (butantoxin), potassium channel toxin α-KTx12.3 (butantoxin-like peptide), potassium channel toxin α-KTx15.1 (peptide Aa1), potassium channel toxin α-KTx15.3 (toxin AmmTX3), potassium channel toxin α-KTx15.6 (disclepin), potassium channel toxin α-KTx16.1 (tamlotoxin), potassium channel toxin α-KTx19.1 (neurotoxin BmBKTx1), potassium channel toxin α-KTx1.3 (iberiotoxin), potassium channel toxin α-KTx 1.4 (Lymbatoxin), potassium channel toxin α-KTx1.7 (Lqh15-1), potassium channel toxin α-KTx1.9 (Hongotoxin-2), potassium channel toxin α-KTx1.10 (Parabtoxin-3), potassium channel toxin α-KTx1.11 (Slotoxin), potassium channel toxin α-KTx1.13 (Calybudotoxin C), potassium channel toxin α-KTx2.1 (Noxiustoxin), potassium channel toxin α-KTx2.2 (Margatoxin), potassium channel toxin α-KTx2.3 (CllTx1 ), potassium channel toxin α-KTx2.4 (noxyus toxin-2), potassium channel toxin α-KTx2.5 (hongotoxin-1), potassium channel toxin α-KTx2.6 (hongotoxin-3), potassium channel toxin α-KTx2.7 (CllTx2), potassium channel toxin α-KTx2.8 (toxin Ce1), potassium channel toxin α-KTx2.9 (toxin Ce2), potassium channel toxin α-KTx2.10 (toxin Ce3), potassium channel toxin α-KTx2.11 (toxin Ce4), potassium channel toxin α-KTx2.12 (Toxin Ce5), potassium channel toxin α-KTx3.1 (Caryotoxin-1), potassium channel toxin α-KTx3.2 (Agitoxin-2), potassium channel toxin α-KTx3.3 (Agitoxin-3), potassium channel toxin α-KTx3.4 (Agitoxin-1), potassium channel toxin α-KTx3.7 (OsK-1), potassium channel toxin α-KTx3.8 (Calybdotoxin-like peptide Bs6), potassium channel toxin α-KTx3.9 (Caryotoxin-3), potassium channel toxin α-KTx4.1 (Tichutoxin K-α), potassium channel toxin α-KTx4.3 (Toxin TdK1), potassium channel toxin α-KTx4.4 (Toxin Tc30), potassium channel toxin α-KTx5.1 (Raylotoxin-1), potassium channel toxin α-KTx5.2 (Raylotoxin-like toxin P05), potassium channel toxin α-KTx5.4 (Tamapin), potassium channel toxin α-KTx5.5 (Ta Mapin-2), potassium channel toxin α-KTx6.1 (potassium channel blocker toxin 1), potassium channel toxin α-KTx6.2 (maurotoxin), potassium channel toxin α-KTx6.3 (neurotoxin HsTX1), potassium channel toxin α-KTx6.12 (anulocotoxin), potassium channel toxin α-KTx6.13 (spinoxin), potassium channel toxin α-KTx6.14 (HgeTx1), potassium channel toxin α-KTx7.2 ( Potassium channel toxin (PiTX-K-β), potassium channel toxin γ-KTx1.2 (Ergtoxin-like protein 1), potassium channel toxin γ-KTx1.3 (Ergtoxin-like protein 1), potassium channel toxin γ-KTx1.4 (Ergtoxin-like protein 1), potassium channel toxin γ-KTx1.5 (Ergtoxin-like protein 1), potassium channel toxin γ-KTx1.6 (Ergtoxin-like protein 1), potassium channel toxin γ-KTx4.2 (Ergtoxin-like protein 5), Insectotoxin-I1, Smalltoxin (peptide I), Insectotoxin-I3 (BeI3), Insectotoxin-I4 (BeI4), Insectotoxin-I5A, Neurotoxin 8 (Neurotoxin VIII), Presumptive toxin Lqh8 / 6, Neurotoxin 9 (Neurotoxin IX), Maurocalcin (MCa), Chlorotoxin-like peptide Bs14 (Bs14), Chlorotoxin (CTX), Neurotoxin P2, Insectotoxin-I5 (BeI5), Potassium channel toxin α-KTx6.15 (Hemitoxin), Toxin GaTx1, AahIT1, Faodotoxin, BaIT2, BotIT1, BotIT2, BmK M1, BmK-M2, BmK-M4, BmK-M7, BmK IT-AP, Bom3, Bom4, BjaIT, Bj-xtrIT, BjIT2, LqhaIT, Lqhb1, LqhIT2, LqhdprIT3a, Lgh-xtrIT, Lqh3, Lqh6, Lqh7, LqqIT1, LqqIT2, Lqq3, OD1, Ts1, or Tz1. .

[0293] In some embodiments, CRIP may be a scorpion peptide having the amino acid sequence shown in any one of SEQ ID NOs. 88 to 191.

[0294] In some embodiments, CRIP may be imperatoxin, a peptide toxin derived from the venom of the African scorpion (Pandinus imperator).

[0295] In some embodiments, CRIP may be imperatoxin, which is imperatoxin A (IpTx-a) or a variant thereof. In some embodiments, IpTx-a has the amino acid sequence GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR (SEQ ID NO: 66).

[0296] In some embodiments, CRIP may be the AaIT1 toxin. The protein toxin AaIT1 is a sodium channel site 4 toxin derived from the North African desert scorpion (Androctonus australis). An exemplary AaIT1 toxin is a peptide having the amino acid sequence of SEQ ID NO: 88 (NCBI accession number P01497.2). AaIT1 is a site 4 toxin that forces the opening of insect sodium channels by lowering the activation reaction energy barrier.

[0297] In some embodiments, the scorpion peptide may contain an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with SEQ ID NOs. 66, 88-191.

[0298] In some cases, polynucleotides encoding scorpion peptides or toxins may encode scorpion peptides or toxins having amino acid sequences that are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the amino acid sequences shown in Sequence ID No. 66, 88-191.

[0299] Anemone peptides and toxins In some embodiments, CRIP can be isolated from a sea anemone. For example, in some embodiments, the sea anemone may be Actinia equina, Anemonia erythraea, Anemonia sulcata, Anemonia viridis, Anthopleura elegantissima, Anthopleura fuscoviridis, Anthopleura xanthogrammica, Bunodosoma caissarum, Bunodosoma cangicum, Bunodosoma granulifera, Heteractis crispa, Parasicyonis actinostoloides, Radianthus paumotensis, or Stoichactis helianthus. In yet other embodiments, the sea anemone toxin may be Av2, Av3, or a variant thereof.

[0300] In some embodiments, CRIP may be one of the following anemone toxins: toxin AETX-1 (AETX I), toxin APETx1, toxin APETx2, antihypertensive protein BDS-1 (antihypertensive substance I), antihypertensive protein BDS-2 (antihypertensive substance II), neurotoxin Bg-2 (Bg II), neurotoxin Bg-3 (Bg III), toxin APE1-1, toxin APE1-2, neurotoxin 1 (toxin ATX-I), neurotoxin 1 (neurotoxin I), neurotoxin 1 (toxin RTX-I), neurotoxin 1 (toxin SHP-I), toxin APE2-1, toxin APE2-2, neurotoxin-2 (toxin ATX-II), (aka AV2) Neurotoxin-2 (Toxin AFT-II), Neurotoxin 2 (Toxin RTX-II), Neurotoxin 2 (Neurotoxin II), Neurotoxin 3 homolog (Neurotoxin III homolog), Neurotoxin 3 (Toxin RTX-III), Neurotoxin 3 (Neurotoxin-III), Neurotoxin 4 (Toxin RTX-IV), Neurotoxin-5 (Toxin ATX-V), Neurotoxin 5 (Toxin RTX-V), Antproylin-A (Toxin AP-A), Antproylin-B ( Toxin AP-B), antproylin-C (toxin AP-C), potassium channel toxin Aek, potassium channel toxin Bgk, major neurotoxin BcIII, neurotoxin BcIV, cangitoxin (CGTX), potassium channel toxin ShK, toxin PCR1 (PCR1-2), toxin PCR2 (PCR2-5), toxin PCR3 (PCR2-1), toxin PCR4 (PCR2-10), toxin PCR6 (PCR3-7), cangitoxin-2 (cangitoxin II), or cangitoxin-3 (cangitoxin III).

[0301] In some embodiments, CRIP may be an anemone peptide having the amino acid sequences shown in SEQ ID NOs. 371-411.

[0302] In some embodiments, the CRIP of the present invention may be one or more polypeptides derived from the sea anemone Anemonia viridis, each possessing a variety of toxins used to defend itself. One of the toxins derived from Anemonia viridis is the neurotoxin "Av3". Av3 is a voltage-gated sodium (Na) receptor at receptor site 3. + Av3 is a type III anemone toxin that inhibits the inactivation of the ) channel, resulting in contractile paralysis. When Av3 toxin binds to site 3, the sodium channel becomes inactivated and destabilized, and then the channel remains in the open position (see Blumenthal et al., Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochem Biophys. 2003;38(2):215-38). Av3 shows high selectivity for sodium channels in crustaceans and insects, and low selectivity for sodium channels in mammals (see Moran et al., Sea anemone toxins affecting voltage-gated sodium channels - molecular and evolutionary features. Toxicon. 2009 Dec 15;54(8):1089-1101). An exemplary Av3 polypeptide from Anemonia viridis having the amino acid sequence of SEQ ID NO: 44 is provided.

[0303] In some embodiments, the CRIP of the present invention may be an Av3 variant polypeptide (AVP). In some embodiments, AVP may have the following amino acid variations from SEQ ID NO: 44: N-terminal amino acid substitution of R1K compared to SEQ ID NO: 44 (modification of polypeptide sequence from wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "KSCCPCYWGGCPWGQNCYPEGCSGPKV" (SEQ ID NO: 45)); C-terminal amino acid deletion compared to SEQ ID NO: 44 (modification of polypeptide sequence from wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "RSCCPCYWGGCPWGQNCYPEGCSGPK" (SEQ ID NO: 46)); and / or N-terminal and C-terminal mutations (modification of polypeptide sequence from wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "KSCCPCYWGGCPWGQNCYPEGCSGPK" (SEQ ID NO: 47), where the N-terminal amino acid may have an R1K substitution compared to SEQ ID NO: 44, and the C-terminal amino acid may be deleted compared to SEQ ID NO: 44).

[0304] In some embodiments, exemplary Av3 peptides or their variants are described in the applicant's PCT application filed on 13 September 2019, entitled "Av3 Mutant Insecticidal Polypeptide and Methods for Manufacturing and Using the Same" (International Application PCT / US19 / 51093), which contains the disclosures thereof and of Av3 peptides or their variants, and is incorporated herein by reference in their entirety.

[0305] In some embodiments, the anemone peptide may contain amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with SEQ ID NOs.

[0306] In some embodiments, the polynucleotide encoding the anemone peptide is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, and at least 88% identical to the amino acid sequences shown in SEQ ID NOs. It is possible to encode an anemone peptide having an amino acid sequence that is at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical.

[0307] Cone snail peptides and conotoxins Conotoxins are toxins isolated from cone snails that act by interfering with neuronal communication. Examples of conotoxins include α-, ω-, μ-, δ-, and κ-conotoxins. Briefly, α-conotoxin (and αA- and φ-conotoxins) targets nicotinic ligand-gated channels, ω-conotoxin targets voltage-gated calcium channels, μ-conotoxin targets voltage-gated sodium channels, δ-conotoxin targets voltage-gated sodium channels, and κ-conotoxin targets voltage-gated potassium channels.

[0308] In some embodiments, CRIP can be isolated from organisms belonging to the genus Cone snails, and the isolated peptide is a conotoxin.

[0309] In some embodiments, CRIP is isolated from Conus amadis, Conus catus, Conus ermineus, Conus geographus, Conus gloriamaris, Conus kinoshitai, Conus magus, Conus marmoreus, Conus purpurascens, Conus stercusmuscarum, Conus striatus, Conus textile, or Conus tulipa.

[0310] Other CRIP In some embodiments, CRIP may be a toxin, peptide, or protein (also known as venom or venom peptide or protein) produced and / or isolated from arthropods, spiders, scorpions, insects, bees, wasps, centipedes, crustaceans, reptiles, snakes, lizards, amphibians, frogs, salamanders, mollusks, cone snails, cnidarians, sea anemones, jellyfish, hydroids, cephalopods, octopuses, squid, cuttlefish, fish, or mammals.

[0311] In some embodiments, CRIP may be snake venom or a toxin derived therefrom.

[0312] CRIP - Insecticide Protein A CRIP-insecticide protein is any protein, peptide, polypeptide, amino acid sequence, composition, or arrangement comprising: (1) at least one CRIP or two or more CRIPs, and (2) additional non-CRIP peptides, polypeptides, or proteins (for example, having the ability in some embodiments to: increase mortality and / or inhibit insect growth when insects are exposed to the CRIP-insecticide protein compared to CRIP alone; increase the expression of the CRIP-insecticide protein, for example in host cells or expression systems; and / or affect the post-translational processing of the CRIP-insecticide protein).

[0313] In some embodiments, the CRIP-insecticide protein may be a polymer containing two or more CRIPs. In some embodiments, the CRIP-insecticide protein may be a polymer containing two or more CRIPs, in which the CRIPs are operably linked via linker peptides (e.g., cleavable and / or non-cleavable linkers).

[0314] In some embodiments, CRIP-insecticide proteins may refer to one or more CRIPs operably linked to one or more proteins such as a stabilizing domain (STA), an endoplasmic reticulum signaling protein (ERSP), an insect-cuttable or insect-incuttable linker (L), and / or any other combination thereof.

[0315] In some embodiments, the CRIP-insecticide protein may be a protein that does not exist in nature, comprising (1) wild-type CRIP and (2) an additional peptide, polypeptide, or protein (e.g., ERSP, linker, STA, UBI, or histidine tag or similar marker).

[0316] In some embodiments, the CRIP-insecticide protein may be a protein that does not exist in nature, comprising (1) wild-type CRIP and (2) CRIP that does not exist in nature.

[0317] In some embodiments, the CRIP-insecticide protein may be a protein that does not exist in nature, comprising (1) wild-type CRIP, (2) a CRIP that does not exist in nature, and (3) an additional peptide, polypeptide, or protein (e.g., ERSP, linker, STA, UBI, or histidine tag or similar marker).

[0318] In some embodiments, the CRIP-insecticide protein may include any of the CRIPs described herein.

[0319] In some embodiments, the insecticidal protein may include one or more CRIPs disclosed herein. In some embodiments, the insecticidal protein may include a CRIP homopolymer (e.g., two or more CRIP monomers having the same CRIP). In some embodiments, the insecticidal protein may include a CRIP heteropolymer (e.g., two or more CRIP monomers having different CRIP monomers).

[0320] In some embodiments, the insecticidal protein may comprise a fusion protein containing two or more CRIPs separated by a cleavable or non-cleavable linker, the amino acid sequences of each CRIP may be the same or different.

[0321] In some embodiments, the insecticidal protein may comprise a fusion protein containing two or more CRIPs separated by a cleavable or non-cleavable linker, the amino acid sequences of each CRIP may be the same or different, and the linker may be cleavable in the insect's intestine or hemolymph.

[0322] In some embodiments, the insecticidal protein may comprise a fusion protein containing two or more CRIPs separated by a cleavable or non-cleavable linker, the amino acid sequences of each CRIP may be the same or different, and the linker is cleavable in the mammalian intestine.

[0323] Exemplary methods for generating cleavable and non-cleavable linkers can be found in U.S. Patent Application No. 15 / 727,277 and International Application PCT / US2013 / 030042 (their disclosures are incorporated herein by reference in their entirety).

[0324] Method for producing CRIP or peptide-IA Methods for producing proteins are well known in this art, and various techniques are available. For example, in some embodiments, proteins may be produced using recombinant methods or chemically synthesized. This disclosure provides methods for producing CRIP, CRIP-insecticide proteins, and other peptide insecticides (peptides-IAs). These methods are described in detail below.

[0325] In some embodiments, the CRIP of the present invention can be prepared using any known method for producing proteins. For example, in some embodiments, CRIP can be prepared using a recombinant expression system, such as a yeast expression system or a bacterial expression system, but is not limited to these. However, those skilled in the art will recognize that other methods of protein production are available.

[0326] In some embodiments, the present invention provides a method for producing CRIP using a recombinant expression system.

[0327] In some embodiments, the present invention includes, essentially consists of, or comprises a method for producing CRIP, the method comprising (a) preparing a vector comprising a first expression cassette comprising, essentially consisting of, or comprising a polynucleotide or complementary nucleotide sequence thereof that is operable to encode CRIP; (b) introducing the vector into a host cell (e.g., a bacterial or yeast, or an insect, or a plant, or an animal cell); and (c) growing a yeast strain in a culture medium under conditions operable to enable the expression of CRIP and its secretion into the culture medium. In some related embodiments, the host cell is a yeast cell.

[0328] The present invention can be implemented in a wide variety of host cells (see the section on host cells below). In fact, end users of the present invention can implement the teachings in any host cell of their choice. Thus, in some embodiments, the host cell may be any host cell that meets the end user's requirements, i.e., in some embodiments, CRIP expression can be achieved using various host cells and based on the teachings herein. For example, in some embodiments, the user may want to use a particular type of host cell (e.g., yeast cells or bacterial cells) as opposed to another, and a given host cell preference may range from availability to cost.

[0329] For example, in some embodiments, the present invention includes, essentially consists of, or comprises a method for producing CRIP, the method comprising (a) preparing a vector comprising, essentially consisting of, or comprising a first expression cassette comprising a polynucleotide or complementary nucleotide sequence thereof that is operable to encode CRIP; (b) introducing the vector into a host cell (e.g., a bacterium or yeast, or an insect, or a plant, or an animal cell); and (c) growing a yeast strain in a culture medium under conditions operable to enable the expression of CRIP and its secretion into the culture medium. In some relevant embodiments, the host cell is a yeast cell.

[0330] Isolation and mutagenesis of wild-type CRIP CRIP or peptide-peptide insecticides (peptide-IA) can be obtained directly from the source (e.g., by isolating the CRIP or peptide-IA from an animal). Mutant CRIP or peptide-IA can be produced by creating mutations in the wild-type CRIP or peptide-IA polynucleotide sequence, inserting the CRIP or peptide-IA polynucleotide sequence into a suitable vector, transforming the host organism in a manner that expresses the polynucleotide encoding the CRIP or peptide-IA, culturing the host organism to produce a desired amount of CRIP or peptide-IA, and then purifying the CRIP or peptide-IA from the host organism and / or its vicinity.

[0331] The generation of mutations in wild-type CRIP or peptide-IA polynucleotide sequences can be achieved by various means well known to those skilled in the art. Mutagenesis methods include the Kunkel method, cassette mutagenesis, PCR site-directed mutagenesis, "delitto perfetto" techniques, direct gene deletion and site-directed mutagenesis using PCR and a single recyclable marker, direct gene deletion and site-directed mutagenesis using PCR and a single recyclable marker with long homologous regions, the "pop-in pop-out" method, and CRISPR-Cas 9.Exemplary methods of site-directed mutagenesis include: Ruvkun & Ausubel, A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85-8; Wallace et al., Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981 Aug 11;9(15):3647-56; Dalbadie-McFarland et al., Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci US A. 1982 Nov;79(21):6409-13; Bachman, Site-directed mutagenesis. Methods Enzymol. 2013;529:241-8; and Carey et al., PCR-mediated This can be found in site-directed mutagenesis. Cold Spring Harb Protoc. 2013 Aug 1;2013(8):738-42, and Cong et al., Multiplex genome engineering using CRISPR / Cas systems. Science. 2013 Feb 15;339(6121):819-23 (all disclosures in the aforementioned references are incorporated herein by reference in their entirety).

[0332] Wild-type CRIPs, such as spider venom, scorpion venom, and / or other toxins, can be isolated from venomous fluids. For example, spider venom can be isolated from the venom glands of spiders (e.g., spiders such as *Eratigena agrestis*) using any technique known to those skilled in the art. For example, in some embodiments, venom can be isolated from spiders according to the method described in U.S. Patent No. 5,688,764 (the disclosure thereof is incorporated herein by reference in its entirety).

[0333] Wild-type CRIP or peptide-IA polynucleotide sequences can be obtained by screening a genomic library using primer probes targeting CRIP or peptide-IA polynucleotide sequences. Alternatively, CRIP or peptide-IA polynucleotide sequences, and / or mutant CRIP or peptide-IA polynucleotide sequences, can be chemically synthesized. For example, CRIP or peptide-IA polynucleotide sequences, and / or mutant CRIP or peptide-IA polynucleotide sequences, can be produced using oligonucleotide synthesis methods such as phosphoramidite, triester, phosphite, or H-phosphonate methods. See Engels, J. Wand Uhlmann, E. (1989), Gene Synthesis (New Synthetic Methods (77)). Angew. Chem. Int. Ed. Engl., 28:716-734 (the disclosure is incorporated herein by reference in its entirety).

[0334] Chemical synthesis of CRIP or peptide-IA polynucleotides In some embodiments, polynucleotide sequences encoding CRIP or peptide-IA may be chemically synthesized using commercially available polynucleotide synthesis services, such as those provided by GENEWIZ® (e.g., TurboGENE®; PriorityGENE, and FragmentGENE) or SIGMA-ALDRICH® (e.g., custom DNA and RNA oligo design and custom DNA oligo ordering). Exemplary methods for producing DNA and / or custom chemically synthesized polynucleotides are well known in the art and are provided exemplarily in U.S. Patent No. 5,736,135 (08 / 389,615), filed February 13, 1995 (the disclosure thereof is incorporated herein by reference in its entirety).Also, Agarwal, et al., Chemical synthesis of polynucleotides. Angew Chem Int Ed Engl.1972 Jun;11(6):451-9, Ohtsuka et al., Recent developments in the chemical synthesis of polynucleotides. Nucleic Acids Res.1982 Nov 11;10(21):6553-6570, Sondek & Shortle.A general strategy for random insertion and substitution. mutagenesis:substoichiometric coupling of trinucleotide phosphoramidites.Proc Natl Acad Sci US A.1992 Apr 15;89(8):3581-3585, Beaucage SL,et al.,Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach.Tetrahedron,Elsevier Science See also Publishers, Amsterdam, NL, vol.48, No.12, 1992, pp.2223-2311, and Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties; Methods in Molecular Biology Vol.20 (the disclosure thereof is incorporated herein by reference in its entirety).

[0335] Chemically synthesized polynucleotides allow for the creation of DNA sequences that are tuned to produce desired polypeptides based on the arrangement of nucleotides within the sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A], or thymine [T] molecules), and mRNA sequences transcribed from chemically synthesized DNA polynucleotides can be translated into amino acid sequences of each amino acid corresponding to the codons in the mRNA sequence. Thus, the amino acid composition of the polypeptide chain translated from the mRNA sequence can be altered by changing the underlying codon that determines which of the 20 amino acids is added to the elongated polypeptide. Therefore, mutations in DNA, such as insertions, substitutions, deletions, and frameshifts, can result in amino acid insertions, substitutions, or deletions depending on the underlying codon.

[0336] Obtaining CRIP or peptide-IA from chemically synthesized DNA polynucleotide sequences and / or wild-type DNA polynucleotide sequences modified via mutagenesis can be achieved by cloning the DNA sequence into a suitable vector. A variety of expression vectors, host organisms, and cloning strategies known to those skilled in the art are available. For example, the vector may be a plasmid, into which a heterologous gene and / or expression cassette to be transcribed and translated can be introduced into yeast cells. The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted and introduced into a cell into which it can replicate. A vector may contain “vector elements” such as an origin of replication (ORI), a gene conferring antibiotic resistance to enable selection, multiple cloning sites, a promoter region, a selection marker for non-bacterial transfection, and a primer binding site. A nucleic acid sequence may be “exogenous,” meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell but is located in a position within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well equipped to construct vectors through standard recombination techniques described in Sambrook et al., 1989 and Ausubel et al., 1996 (both incorporated herein by reference). In addition to encoding CRIPs or peptide-IA polynucleotides, vectors can encode targeted molecules. Target molecules guide desired nucleic acids to specific tissues, cells, or other locations.

[0337] Vectors and transformations In some embodiments, CRIP or peptide-IA polynucleotides can be cloned into vectors using various cloning strategies and commercially available cloning kits and materials readily available to those skilled in the art. For example, CRIP or peptide-IA polynucleotides can be cloned into vectors using strategies (e.g., SnapFast, Gateway, TOPO, Gibson, LIC, InFusionHD, or Electra strategies). Numerous vectors are commercially available and can be used to produce CRIP or peptide-IA. For example, CRIP or peptide-IA polynucleotides can be generated at room temperature for 5 minutes using polymerase chain reaction (PCR) in combination with the pCR®II-TOPO vector or the PCR®2.1-TOPO® vector (commercially available as the TOPO®TA Cloning® Kit from Invitrogen). The TOPO® reaction product can then be used to transform competent cells, which can then be selected based on color changes (see Janke et al., A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug;21(11):947-62; also see Adams et al., Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997. This disclosure is incorporated herein by reference in its entirety).

[0338] In some embodiments, the polynucleotide encoding CRIP or peptide-IA may be cloned into vectors such as plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and / or artificial chromosomes (e.g., YAC).

[0339] In some embodiments, a polynucleotide encoding CRIP or peptide-IA can be inserted into a vector (e.g., a plasmid vector) using E. coli as the host by: digesting approximately 2–5 μg of vector DNA with the necessary restriction enzymes to enable insertion of the desired DNA segment, followed by overnight incubation to achieve complete digestion (the 5' end can be dephosphorylated using alkaline phosphatase to avoid autoligation / recyclization); purifying the digested vector with a gel; amplifying the desired DNA segment (e.g., the polynucleotide encoding CRIP or peptide-IA) via PCR, and removing any excess enzymes, primers, unincorporated dNTPs, incompletely lengthy failed PCR products, and / or salts from the PCR reaction using techniques known to those skilled in the art (e.g., by using a PCR cleanup kit). A mixture is prepared containing approximately 20 ng of vector, approximately 100-1,000 ng or the target DNA segment, 2 μL of 10× buffer (i.e., 30 mM Tris-HCl, 4 mM MgCl2, 26 μM NAD, 1 mM DTT, 50 μg / ml BSA, pH 8; stored at 25°C), and 1 μL of T4 DNA ligase. The target DNA segment is ligated into the vector by adding H2O to bring the total volume to 20 μL. The ligation reaction mixture can then be incubated at room temperature for 2 hours or overnight at 16°C. The ligation reaction mixture (i.e., approximately 1 μL) can then be used to transform competent cells, for example, by electroporation or chemical methods. Colony PCR can then be performed to identify the vector containing the target DNA segment.

[0340] In some embodiments, a polynucleotide encoding CRIP or peptide-IA, along with other DNA segments, can constitute a CRIP or peptide-IA expression ORF designed for secretion from host yeast cells. An exemplary method for designing a CRIP or peptide-IA expression ORF is as follows: The ORF may begin with a signal peptide sequence, followed by a DNA sequence encoding a Kex2 cleavage site (lysine-arginine), and then a CRIP or peptide-IA polynucleotide transgene (with a glycine-serine codon at the 5' end and a stop codon at the 3' end). All these elements are then expressed in yeast cells as a single open reading frame (ORF) in the fusion peptide. The α-conjugation factor (αMF) signaling sequence is most frequently used to facilitate the metabolic process of recombinant insecticidal peptides via the endogenous secretory pathway in recombinant yeast. Specifically, the expressed fusion peptide typically enters the endoplasmic reticulum, where the α-conjugation factor signaling sequence is removed by signal peptidase activity. The resulting insecticidal propeptide is then transported to the Golgi apparatus, where the aforementioned lysine-arginine dipeptide is completely removed by Kex2 endoprotease, after which the mature polypeptide (i.e., CRIP or peptide-IA) is secreted from the cell.

[0341] In some embodiments, polypeptide expression levels in recombinant yeast cells can be enhanced by optimizing codons based on a specific host yeast species. The naturally occurring codon frequencies observed in the endogenous open reading frame of a given host organism do not necessarily need to be optimized for high-efficiency expression. Furthermore, different yeast species (e.g., Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.) have different optimal codons for high-efficiency expression. Therefore, for CRIP or peptide-IA expression ORFs containing sequence elements encoding signal sequences, Kex2 cleavage sites, and CRIP or peptide-IA, codon optimization should be considered because they are initially translated as a single fusion peptide in recombinant yeast cells.

[0342] In some embodiments, a codon-optimized CRIP or peptide-IA expression ORF can be ligated into a yeast-specific expression vector for yeast expression. Many expression vectors are available for yeast expression, including episomal vectors and insertion vectors, which are typically designed for specific yeast strains. Careful selection of an appropriate expression vector is necessary, taking into account the specific yeast expression system used for peptide production. In some embodiments, an insertion vector can be used, which is integrated into the chromosome of a transformed yeast cell and maintained stably throughout the cell division and proliferation cycle. The insertion DNA sequence is homologous to the target genomic DNA locus in the transformed yeast species, and such insertion sequences include pLAC4, 25S rDNA, pAOX1, and TRP2. The location of the insecticidal peptide transgene may be adjacent to the insertion DNA sequence (insertion vector) or within the insertion DNA sequence (substitution vector).

[0343] In some embodiments, the expression vector may include E. coli elements for DNA preparation in E. coli (e.g., E. coli replication origin, antibiotic selection marker, etc.). In some embodiments, the vector may include a set of sequence elements necessary for the expression of the target transgene (e.g., transcription promoter, terminator, yeast selection marker, integrated DNA sequence homologous to host yeast DNA, etc.). Many suitable yeast promoters exist, including natural and engineered promoters (e.g., yeast promoters such as pLAC4, pAOX1, pUPP, pADH1, pTEF, pGal1, and others), and can be used in some embodiments.

[0344] In some embodiments, selection methods such as acetamide prototrophic selection, zeosin resistance selection, genetisin resistance selection, knowureotricin resistance selection, uracil deletion selection, and / or other selection methods may be used. For example, in some embodiments, the amdS gene of Aspergillus nidulans can be used as a selection marker. Exemplary methods for using selection markers can be found in U.S. Patent No. 6,548,285 (filed April 3, 1997), No. 6,165,715 (filed June 22, 1998), and No. 6,110,707 (filed January 17, 1997) (their disclosures are incorporated herein by reference in their entirety).

[0345] In some embodiments, a polynucleotide encoding CRIP or peptide-IA may be inserted into the pKLAC1 plasmid. pKLAC1 is commercially available from New England Biolabs® Inc. (item number (NEB#E1000)). pKLAC1 is designed to achieve high levels of recombinant protein expression (e.g., CRIP or peptide-IA) in the yeast Kluyveromyces lactis. The pKLAC1 plasmid can be ordered alone or as part of a K. lactis protein expression kit. The pKLAC1 plasmid can be linearized using SacII or BstXI restriction enzymes and has an MCS downstream of the αMF secretion signal. The αMF secretion signal leads the recombinant protein into the secretory pathway, where it is then cleaved by Kex2 to obtain, for example, CRIP or peptide-IA. Kex2 is a calcium-dependent serine protease involved in the activation of proproteins in the secretory pathway and is commercially available (PeproTech®; item numbers 450-45).

[0346] In some embodiments, following the selection of yeast colonies transformed with a pKLAC1 plasmid ligated with a polynucleotide encoding CRIP or peptide-IA, the polynucleotide encoding CRIP or peptide-IA can be inserted into or subcloned into the pKlac1 plasmid. Yeast (e.g., K. lactis) transformed with a pKLAC1 plasmid ligated with a polynucleotide encoding CRIP or peptide-IA may be selected based on acetamidase (amdS), which allows the transformed yeast cells to grow in YCB medium containing acetamide as its sole nitrogen source. Identify positive yeast colonies transformed with a pKLAC1 plasmid ligated with a polynucleotide encoding CRIP or peptide-IA.

[0347] In some embodiments, the polynucleotide encoding CRIP or peptide-IA can be inserted into other commercially available plasmids and / or vectors readily available to those skilled in the art, for example, plasmids are available from Addgene (a non-profit plasmid repository), GenScript®, Takara®, Qiagen®, and Promega®.

[0348] In some embodiments, the polynucleotide encoding the TVP can be inserted into other commercially available plasmids and / or vectors readily available to those skilled in the art, for example, plasmids available from Addgene (a non-profit plasmid repository), GenScript®, Takara®, Qiagen®, and Promega®.

[0349] In some embodiments, yeast cells transformed with one or more CRIP expression cassettes are given CRIP in yeast culture at concentrations of at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, and at least 190 mg / L per liter of medium. 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, less It can be produced in a yield of CRIP of at least 11,000 mg / L, at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L.

[0350] In some embodiments, one or more expression cassettes containing polynucleotides capable of expressing CRIP are inserted into the vector, resulting in CRIP concentrations of approximately 100 mg / L to 100,000 mg / L, 110 mg / L to 100,000 mg / L, 120 mg / L to 100,000 mg / L, 130 mg / L to 100,000 mg / L, 140 mg / L to 100,000 mg / L, 150 mg / L to 100,000 mg / L, 160 mg / L to 100,000 mg / L, and 170 mg / L per liter of culture medium (supernatant of yeast fermentation). L ~ about 100,000mg / L, about 180mg / L - about 100,000mg / L, about 190mg / L - about 100,000mg / L, about 200mg / L - about 100,000mg / L, about 250mg / L - about 100,000mg / L, about 500mg / L - about 100,000mg / L , about 750mg / L to about 100,000mg / L, about 1000mg / L to about 100,000mg / L, about 1000mg / L to about 100,000mg / L, about 1500mg / L to about 100,000mg / L, about 2000mg / L to about 100,000mg / L, about 2500mg / L ~100,000mg / L, 3000mg / L~100,000mg / L, 3500mg / L~100,000mg / L, 4000mg / L~100,000mg / L, 4500mg / L~100,000mg / L, 5000mg / L~100,000 mg / L, about 5500mg / L to about 100,000mg / L, about 6000mg / L to about 100,000mg / L, about 6500mg / L to about 100,000mg / L, about 7000mg / L to about 100,000mg / L, about 7500mg / L to about 100,000mg / L, about 800 0mg / L~about 100,000mg / L, about 8500mg / L~about 100,000mg / L, about 9000mg / L~about 100,000mg / L, about 9500mg / L~about 100,000mg / L, about 10000mg / L~about 100,000mg / L, about 10500mg / L~about 100,000mg / L, about 11000mg / L~about 100,000mg / L, about 11500mg / L~about 100,000mg / L, about 12000mg / L~about 100,000mg / L, about 12500mg / L~about 100,000mg / L, about 13000mg / L~about 100,000mg / L, approximately 13500mg / L to approximately 100,000mg / L, approximately 14000mg / L to approximately 100,000mg / L, approximately 14500mg / L to approximately 100,000mg / L, approximately 15000mg / L to approximately 100,000mg / L, approximately 15500mg / L to approximately 100,000mg / L, approximately 16000mg / L to approximately 100,000mg / L, approximately 16500mg / L to approximately 100,000mg / L, approximately 17000mg / L to approximately 100,000mg / L, approximately 17500mg / L to approximately 100,000mg / L, approximately 18000mg / L to approximately 100,000mg / L L, approximately 18500 mg / L to approximately 100,000 mg / L, approximately 19000 mg / L to approximately 100,000 mg / L, approximately 19500 mg / L to approximately 100,000 mg / L, approximately 20000 mg / L to approximately 100,000 mg / L, approximately 20500 mg / L to approximately 100,000 mg / L, approximately 21000 mg / L to approximately 100,000 mg / L, approximately 21500 mg / L to approximately 100,000 mg / L, approximately 22000 mg / L to approximately 100,000 mg / L, approximately 22500 mg / L to approximately 100,000 mg / L, approximately 23000 mg / L to approximately 100,000 mg / L, approximately 235 00 mg / L ~ approx. 100,000 mg / L, approx. 24,000 mg / L ~ approx. 100,000 mg / L, approx. 24,500 mg / L ~ approx. 100,000 mg / L, approx. 25,000 mg / L ~ approx. 100,000 mg / L, approx. 25,500 mg / L ~ approx. 100,000 mg / L, approx. 26,000 mg / L ~ approx. 100,000 mg / L, approx. 26,500 mg / L ~ approx. 100,000 mg / L, approx. 27,000 mg / L ~ approx. 100,000 mg / L, approx. 27,500 mg / L ~ approx. 100,000 mg / L, approx. 28,000 mg / L ~ approx. 100,000 mg / L, approx. 28,500 mg / L ~approximately 100,000 mg / L, approximately 29,000 mg / L, approximately 29,500 mg / L, approximately 100,000 mg / L, approximately 30,000 mg / L, approximately 30,500 mg / L, approximately 100,000 mg / L, approximately 31,000 mg / L, approximately 31,500 mg / L, approximately 100,000 mg / L, approximately 32,000 mg / L, approximately 32,500 mg / L, approximately 100,000 mg / L, approximately 33,000 mg / L, approximately 100,000 mg / L, approximately 33,500 mg / L, approximately 100,000mg / L, approximately 34000mg / L to approximately 100,000mg / L, approximately 34500mg / L to approximately 100,000mg / L, approximately 35000mg / L to approximately 100,000mg / L, approximately 35500mg / L to approximately 100,000mg / L, approximately 36000mg / L to approximately 100,000mg / L, approximately 36500mg / L to approximately 100,000mg / L, approximately 37000mg / L to approximately 100,000mg / L, approximately 37500mg / L to approximately 100,000mg / L, approximately 38000mg / L to approximately 100,000mg / L, approximately 38500mg / L to approximately 100,000mg / L L, approximately 39000 mg / L to approximately 100,000 mg / L, approximately 39500 mg / L to approximately 100,000 mg / L, approximately 40000 mg / L to approximately 100,000 mg / L, approximately 40500 mg / L to approximately 100,000 mg / L, approximately 41000 mg / L to approximately 100,000 mg / L, approximately 41500 mg / L to approximately 100,000 mg / L, approximately 42000 mg / L to approximately 100,000 mg / L, approximately 42500 mg / L to approximately 100,000 mg / L, approximately 43000 mg / L to approximately 100,000 mg / L, approximately 43500 mg / L to approximately 100,000 mg / L, approximately 440 00 mg / L ~ approximately 100,000 mg / L, approximately 44,500 mg / L ~ approximately 100,000 mg / L, approximately 45,000 mg / L ~ approximately 100,000 mg / L, approximately 45,500 mg / L ~ approximately 100,000 mg / L, approximately 46,000 mg / L ~ approximately 100,000 mg / L, approximately 46,500 mg / L ~ approximately 100,000 mg / L, approximately 47,000 mg / L ~ approximately 100,000 mg / L, approximately 47,500 mg / L ~ approximately 100,000 mg / L, approximately 48,000 mg / L ~ approximately 100,000 mg / L, approximately 48,500 mg / L ~ approximately 100,000 mg / L, approximately 49,000 mg / L ~approximately 100,000 mg / L, approximately 49,500 mg / L~approximately 100,000 mg / L, approximately 50,000 mg / L~approximately 100,000 mg / L, approximately 50,500 mg / L~approximately 100,000 mg / L, approximately 51,000 mg / L~approximately 100,000 mg / L, approximately 51,500 mg / L~approximately 100,000 mg / L, approximately 52,000 mg / L~approximately 100,000 mg / L, approximately 52,500 mg / L~approximately 100,000 mg / L, approximately 53,000 mg / L~approximately 100,000 mg / L, approximately 53,500 mg / L~approximately 100,000 mg / L, approximately 54,000 mg / L~approximately 100,000mg / L, approximately 54500mg / L to approximately 100,000mg / L, approximately 55000mg / L to approximately 100,000mg / L, approximately 55500mg / L to approximately 100,000mg / L, approximately 56000mg / L to approximately 100,000mg / L, approximately 56500mg / L to approximately 100,000mg / L, approximately 57000mg / L to approximately 100,000mg / L, approximately 57500mg / L to approximately 100,000mg / L, approximately 58000mg / L to approximately 100,000mg / L, approximately 58500mg / L to approximately 100,000mg / L, approximately 59000mg / L to approximately 100,000mg / L L, approximately 59500 mg / L to approximately 100,000 mg / L, approximately 60000 mg / L to approximately 100,000 mg / L, approximately 60500 mg / L to approximately 100,000 mg / L, approximately 61000 mg / L to approximately 100,000 mg / L, approximately 61500 mg / L to approximately 100,000 mg / L, approximately 62000 mg / L to approximately 100,000 mg / L, approximately 62500 mg / L to approximately 100,000 mg / L, approximately 63000 mg / L to approximately 100,000 mg / L, approximately 63500 mg / L to approximately 100,000 mg / L, approximately 64000 mg / L to approximately 100,000 mg / L, approximately 645 00 mg / L ~ approx. 100,000 mg / L, approx. 65,000 mg / L ~ approx. 100,000 mg / L, approx. 65,500 mg / L ~ approx. 100,000 mg / L, approx. 66,000 mg / L ~ approx. 100,000 mg / L, approx. 66,500 mg / L ~ approx. 100,000 mg / L, approx. 67,000 mg / L ~ approx. 100,000 mg / L, approx. 67,500 mg / L ~ approx. 100,000 mg / L, approx. 68,000 mg / L ~ approx. 100,000 mg / L, approx. 68,500 mg / L ~ approx. 100,000 mg / L, approx. 69,000 mg / L ~ approx. 100,000 mg / L, approx. 69,500 mg / L ~approximately 100,000 mg / L, approximately 70,000 mg / L, approximately 70,500 mg / L, approximately 71,000 mg / L, approximately 71,500 mg / L, approximately 72,000 mg / L, approximately 72,500 mg / L, approximately 73,000 mg / L, approximately 73,500 mg / L, approximately 74,000 mg / L, approximately 74,500 mg / L000mg / L, approximately 75000mg / L to approximately 100,000mg / L, approximately 75500mg / L to approximately 100,000mg / L, approximately 76000mg / L to approximately 100,000mg / L, approximately 76500mg / L to approximately 100,000mg / L, approximately 77000mg / L to approximately 100,000mg / L, approximately 77500mg / L to approximately 100,000mg / L, approximately 78000mg / L to approximately 100,000mg / L, approximately 78500mg / L to approximately 100,000mg / L, approximately 79000mg / L to approximately 100,000mg / L, approximately 79500mg / L to approximately 100,000mg / L L, approximately 80,000 mg / L to approximately 100,000 mg / L, approximately 80,500 mg / L to approximately 100,000 mg / L, approximately 81,000 mg / L to approximately 100,000 mg / L, approximately 81,500 mg / L to approximately 100,000 mg / L, approximately 82,000 mg / L to approximately 100,000 mg / L, approximately 82,500 mg / L to approximately 100,000 mg / L, approximately 83,000 mg / L to approximately 100,000 mg / L, approximately 83,500 mg / L to approximately 100,000 mg / L, approximately 84,000 mg / L to approximately 100,000 mg / L, approximately 84,500 mg / L to approximately 100,000 mg / L, approximately 850 00 mg / L ~ approx. 100,000 mg / L, approx. 85,500 mg / L ~ approx. 100,000 mg / L, approx. 86,000 mg / L ~ approx. 100,000 mg / L, approx. 86,500 mg / L ~ approx. 100,000 mg / L, approx. 87,000 mg / L ~ approx. 100,000 mg / L, approx. 87,500 mg / L ~ approx. 100,000 mg / L, approx. 88,000 mg / L ~ approx. 100,000 mg / L, approx. 88,500 mg / L ~ approx. 100,000 mg / L, approx. 89,000 mg / L ~ approx. 100,000 mg / L, approx. 89,500 mg / L ~ approx. 100,000 mg / L, approx. 90,000 mg / L ~approximately 100,000 mg / L, approximately 90,500 mg / L, approximately 91,000 mg / L, approximately 91,500 mg / L, approximately 92,000 mg / L, approximately 92,500 mg / L, approximately 93,000 mg / L, approximately 93,500 mg / L, approximately 94,000 mg / L, approximately 94,500 mg / L, approximately 100,000 mg / L, approximately 95,000 mg / L000mg / L, about 95500mg / L~about 100,000mg / L, about 96000mg / L~about 100,000mg / L, about 96500mg / L~about 100,000mg / L, about 97000mg / L~about 100, 000mg / L, about 97500mg / L~about 100,000mg / L, about 98000mg / L~about 100,000mg / L, about 98500mg / L~about 100,000mg / L, about 99000mg / L~about 100, This can yield a CRIP of 000 mg / L, or in the range of approximately 99,500 mg / L to approximately 100,000 mg / L.

[0351] In some embodiments, one or more expression cassettes containing polynucleotides capable of expressing CRIP are inserted into the vector, resulting in CRIP concentrations of approximately 100 mg / L to 100,000 mg / L, approximately 100 mg / L to 99,500 mg / L, approximately 100 mg / L to 99,000 mg / L, approximately 100 mg / L to 98,500 mg / L, approximately 100 mg / L to 98,000 mg / L, approximately 100 mg / L to 97,500 mg / L, approximately 100 mg / L to 97,000 mg / L, and approximately 100 mg / L to 96,500 mg / L per liter of culture medium (supernatant of yeast fermentation). L, approx. 100 mg / L ~ approx. 96000 mg / L, approx. 100 mg / L ~ approx. 95500 mg / L, approx. 100 mg / L ~ approx. 95000 mg / L, approx. 100mg / L~93000mg / L, 100mg / L~92500mg / L, 100mg / L~92000mg / L, 100mg / L~91500mg / L, 100mg / L~91000mg / L, 100m~90500mg / L, 100m g / L ~ approx. 90000 mg / L, approx. 100 mg / L ~ approx. 89500 mg / L, approx. 100 mg / L ~ approx. 89000 mg / L, approx. 100 mg / L ~ approx. 88500 mg / L, approx. ~87000mg / L, 100mg / L~86500mg / L, 100mg / L~86000mg / L, 100mg / L~85500mg / L, 100mg / L~85000mg / L, 100mg / L~84500mg / L, 100mg / L~84 000mg / L, about 100mg / L to about 83500mg / L, about 100mg / L to about 83000mg / L, about 100mg / L to about 82500mg / L, about 100mg / L to about 82000mg / L, about 100mg / L to about 81500mg / L, about 100mg / L to about 81000m g / L, approx. 100 mg / L ~ approx. 80,500 mg / L, approx. 100 mg / L ~ approx. 80,000 mg / L, approx. 100 mg / L ~ approx. 79,500 mg / L, approx. 100 mg / L ~ approx. 79,000 mg / L, approx.Approximately 100 mg / L to approximately 77500 mg / L, approximately 100 mg / L to approximately 77000 mg / L, approximately 100 mg / L to approximately 76500 mg / L, approximately 100 mg / L to approximately 76000 mg / L, approximately 100 mg / L to approximately 75500 mg / L, approximately 100 mg / L to approximately 75000 mg / L, approximately 100 mg / L to approximately 74500 mg / L, approximately 100 mg / L to approximately 74000 mg / L, approximately 100 mg / L to approximately 73500 mg / L, approximately 100 mg / L to approximately 73000 mg / L, approximately 100 mg / L to approximately 72500 mg / L, approximately 100 mg / L to approximately 72000 mg / L, approximately 100 mg / L to approximately 71500mg / L, approximately 100mg / L to approximately 71000mg / L, approximately 100mg / L to approximately 70500mg / L, approximately 100mg / L to approximately 70000mg / L, approximately 100mg / L to approximately 69500mg / L, approximately 100mg / L to approximately 69000mg / L, approximately 100mg / L to approximately 68500mg / L, approximately 100mg / L to approximately 68000mg / L, approximately 100mg / L to approximately 67500mg / L, approximately 100mg / L to approximately 67000mg / L, approximately 100mg / L to approximately 66500mg / L, approximately 100mg / L to approximately 66000mg / L, approximately 100mg / L to approximately 65500mg / L Approximately 100 mg / L to approximately 65000 mg / L, approximately 100 mg / L to approximately 64500 mg / L, approximately 100 mg / L to approximately 64000 mg / L, approximately 100 mg / L to approximately 63500 mg / L, approximately 100 mg / L to approximately 63000 mg / L, approximately 100 mg / L to approximately 62500 mg / L, approximately 100 mg / L to approximately 62000 mg / L, approximately 100 mg / L to approximately 61500 mg / L, approximately 100 mg / L to approximately 61000 mg / L, approximately 100 mg / L to approximately 60500 mg / L, approximately 100 mg / L to approximately 60000 mg / L, approximately 100 mg / L to approximately 59500 mg / L, approximately 100 mg / L to approximately 59000 mg / L, approximately 100 mg / L to approximately 58500 mg / L, approximately 100 mg / L to approximately 58000 mg / L, approximately 100 mg / L to approximately 57500 mg / L, approximately 100 mg / L to approximately 57000 mg / L, approximately 100 mg / L to approximately 56500 mg / L, approximately 100 mg / L to approximately 56000 mg / L, approximately 100 mg / L to approximately 55500 mg / L, approximately 100 mg / L to approximately 55000 mg / L, approximately 100 mg / L to approximately 54500 mg / L, approximately 100 mg / L to approximately 54000 mg / L, approximately 100 mg / L to approximately 53500 mg / L, approximately 100 mg / L to approximately 53000 mg / LApproximately 100 mg / L to approximately 52500 mg / L, approximately 100 mg / L to approximately 52000 mg / L, approximately 100 mg / L to approximately 51500 mg / L, approximately 100 mg / L to approximately 51000 mg / L, approximately 100 mg / L to approximately 50500 mg / L, approximately 100 mg / L to approximately 50000 mg / L, approximately 100 mg / L to approximately 49500 mg / L, approximately 100 mg / L to approximately 49000 mg / L, approximately 100 mg / L to approximately 48500 mg / L, approximately 100 mg / L to approximately 48000 mg / L, approximately 100 mg / L to approximately 47500 mg / L, approximately 100 mg / L to approximately 47000 mg / L, approximately 100 mg / L to approximately 46500 mg / L, approximately 100 mg / L to approximately 46000 mg / L, approximately 100 mg / L to approximately 45500 mg / L, approximately 100 mg / L to approximately 45000 mg / L, approximately 100 mg / L to approximately 44500 mg / L, approximately 100 mg / L to approximately 44000 mg / L, approximately 100 mg / L to approximately 43500 mg / L, approximately 100 mg / L to approximately 43000 mg / L, approximately 100 mg / L to approximately 42500 mg / L, approximately 100 mg / L to approximately 42000 mg / L, approximately 100 mg / L to approximately 41500 mg / L, approximately 100 mg / L to approximately 41000 mg / L, approximately 100 mg / L to approximately 40500 mg / L Approximately 100 mg / L to approximately 40000 mg / L, approximately 100 mg / L to approximately 39500 mg / L, approximately 100 mg / L to approximately 39000 mg / L, approximately 100 mg / L to approximately 38500 mg / L, approximately 100 mg / L to approximately 38000 mg / L, approximately 100 mg / L to approximately 37500 mg / L, approximately 100 mg / L to approximately 37000 mg / L, approximately 100 mg / L to approximately 36500 mg / L, approximately 100 mg / L to approximately 36000 mg / L, approximately 100 mg / L to approximately 35500 mg / L, approximately 100 mg / L to approximately 35000 mg / L, approximately 100 mg / L to approximately 34500 mg / L, approximately 100 mg / L to approximately 34000 mg / L, approximately 100 mg / L to approximately 33500 mg / L, approximately 100 mg / L to approximately 33000 mg / L, approximately 100 mg / L to approximately 32500 mg / L, approximately 100 mg / L to approximately 32000 mg / L, approximately 100 mg / L to approximately 31500 mg / L, approximately 100 mg / L to approximately 31000 mg / L, approximately 100 mg / L to approximately 30500 mg / L, approximately 100 mg / L to approximately 30000 mg / L, approximately 100 mg / L to approximately 29500 mg / L, approximately 100 mg / L to approximately 29000 mg / L, approximately 100 mg / L to approximately 28500 mg / L, approximately 100 mg / L to approximately 28000 mg / LApproximately 100 mg / L to approximately 27500 mg / L, approximately 100 mg / L to approximately 27000 mg / L, approximately 100 mg / L to approximately 26500 mg / L, approximately 100 mg / L to approximately 26000 mg / L, approximately 100 mg / L to approximately 25500 mg / L, approximately 100 mg / L to approximately 25000 mg / L, approximately 100 mg / L to approximately 24500 mg / L, approximately 100 mg / L to approximately 24000 mg / L, approximately 100 mg / L to approximately 23500 mg / L, approximately 100 mg / L to approximately 23000 mg / L, approximately 100 mg / L to approximately 22500 mg / L, approximately 100 mg / L to approximately 22000 mg / L, approximately 100 mg / L~approx. 21500mg / L, approx. 100mg / L~approx. 21000mg / L, approx. 100mg / L~approx. 20500mg / L, approx. 100mg / L~approx. 20000mg / L, approx. 100mg / L~approx. 19500mg / L, approx. 100mg / L~approx. 19000mg / L, approx. 100mg / L~approx. 18500mg / L, approx. 100mg / L~approx. 18000mg / L, approx. 100mg / L~approx. 17500mg / L, approx. 100mg / L~approx. 17000mg / L, approx. 100mg / L~approx. 16500mg / L, approx. 100mg / L~approx. 16000mg / L, approx. 100mg / L~approx. 155 00 mg / L, approximately 100 mg / L to approximately 15000 mg / L, approximately 100 mg / L to approximately 14500 mg / L, approximately 100 mg / L to approximately 14000 mg / L, approximately 100 mg / L to approximately 13500 mg / L, approximately 100 mg / L to approximately 13000 mg / L, approximately 100 mg / L to approximately 12500 mg / L, approximately 100 mg / L to approximately 12000 mg / L, approximately 100 mg / L to approximately 11500 mg / L, approximately 100 mg / L to approximately 11000 mg / L, approximately 100 mg / L to approximately 10500 mg / L, approximately 100 mg / L to approximately 10000 mg / L, approximately 100 mg / L to approximately 9500 mg / L, Approximately 100 mg / L to approximately 9000 mg / L, approximately 100 mg / L to approximately 8500 mg / L, approximately 100 mg / L to approximately 8000 mg / L, approximately 100 mg / L to approximately 7500 mg / L, approximately 100 mg / L to approximately 7000 mg / L, approximately 100 mg / L to approximately 6500 mg / L, approximately 100 mg / L to approximately 6000 mg / L, approximately 100 mg / L to approximately 5500 mg / L, approximately 100 mg / L to approximately 5000 mg / L, approximately 100 mg / L to approximately 4500 mg / L, approximately 100 mg / L to approximately 4000 mg / L, approximately 100 mg / L to approximately 3500 mg / L, approximately 100 mg / L to approximately 3000 mg / L.This can yield CRIP in the following ranges: approximately 100 mg / L to 2500 mg / L, approximately 100 mg / L to 2000 mg / L, approximately 100 mg / L to 1500 mg / L, approximately 100 mg / L to 1000 mg / L, approximately 100 mg / L to 1000 mg / L, approximately 100 mg / L to 750 mg / L, approximately 100 mg / L to 500 mg / L, approximately 100 mg / L to 250 mg / L, approximately 100 mg / L to 100 mg / L, or approximately 100 mg / L to 110 mg / L.

[0352] In addition to the DNA polynucleotide sequence encoding CRIP or peptide-IA, additional DNA segments known as regulatory elements can be cloned into vectors that enable enhanced expression of foreign DNA or transgenes. Examples of such additional DNA segments include (1) promoter, terminator, and / or enhancer elements, (2) appropriate mRNA-stabilizing polyadenylation signals, (3) internal ribosome entry sites (IRESs), (4) introns, and (5) post-transcriptional regulatory elements. The combination of the target DNA segment and any one of the aforementioned cis-acting elements is called an "expression cassette."

[0353] A single expression cassette may include one or more of the aforementioned regulatory elements and a polynucleotide capable of expressing CRIP or peptide-IA. For example, in some embodiments, a CRIP or peptide-IA expression cassette may include a polynucleotide capable of expressing CRIP or peptide-IA and an α-MF signal, a Kex2 site, an LAC4 terminator, an ADN1 promoter, and an acetamidase (amdS) selection marker adjacent to the LAC4 promoter at the 5' and 3' ends.

[0354] In some embodiments, there may be multiple expression cassettes cloned into the vector. For example, in some embodiments, there may be a first expression cassette containing a polynucleotide capable of expressing CRIP or peptide-IA. In alternative embodiments, there may be two expression cassettes capable of encoding CRIP or peptide-IA (i.e., a dual expression cassette). In other embodiments, there may be three expression cassettes capable of encoding CRIP or peptide-IA (i.e., a triple expression cassette).

[0355] In some embodiments, a dual expression cassette can be generated by subcloning a second CRIP or peptide-IA expression cassette into a vector containing a first CRIP or peptide-IA expression cassette.

[0356] In some embodiments, a triple expression cassette can be generated by subcloning a third CRIP or peptide-IA expression cassette into a vector containing the first and second CRIP or peptide-IA expression cassettes.

[0357] In some embodiments, yeast cells transformed with one or more CRIP or peptide-IA expression cassettes are given CRIP in yeast culture at concentrations of at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, and at least 190 mg / L per liter of medium. 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg CRIP or peptide-IA can be produced in yields of CRIP or peptide-IA of at least 12,000 mg / L, at least 12,500 mg / L, at least 13,000 mg / L, at least 14,000 mg / L, at least 15,000 mg / L, at least 16,000 mg / L, at least 17,000 mg / L, at least 17,500 mg / L, at least 18,000 mg / L, at least 19,000 mg / L, at least 20,000 mg / L, at least 25,000 mg / L, at least 30,000 mg / L, at least 40,000 mg / L, at least 50,000 mg / L, at least 60,000 mg / L, at least 70,000 mg / L, at least 80,000 mg / L, at least 90,000 mg / L, or at least 100,000 mg / L.

[0358] In some embodiments, one or more expression cassettes containing polynucleotides capable of expressing CRIP or peptide-IA can be inserted into a vector (e.g., pKlac1 plasmid) to yield approximately 100 mg / L of CRIP or peptide-IA (supernatant of yeast ferment). For example, in some embodiments, two expression cassettes containing polynucleotides capable of expressing CRIP or peptide-IA can be inserted into a vector (e.g., pKS482 plasmid) to yield approximately 2 g / L of CRIP or peptide-IA (supernatant of yeast ferment). Alternatively, in some embodiments, three expression cassettes containing polynucleotides capable of expressing CRIP or peptide-IA may be inserted into a vector (e.g., pKlac1T plasmid).

[0359] In some embodiments, multiple CRIP or peptide-IA expression cassettes can be transfected into yeast to enable the incorporation of one or more copies of an optimized CRIP or peptide-IA transgene into the K. lactis genome. An exemplary method for introducing multiple CRIP or peptide-IA expression cassettes into the K. lactis genome is as follows: Synthesize a DNA sequence for a CRIP or peptide-IA expression cassette comprising an intact LAC4 promoter element, a codon-optimized CRIP or peptide-IA expression ORF element, and a pLAC4 terminator element. Ligate the intact expression cassette between the Sal I and Kpn I restriction sites of the pKlac1 vector, downstream of the pLAC4 terminator of pKS477, to obtain a dual transgene CRIP or peptide-IA expression vector pKS482. The dual transgene vector pKS482 is then linearized using a Sac II restriction endonuclease and transformed into the K. lactis YCT306 strain by electroporation. Next, the obtained yeast colonies are grown on YCB agar plates supplemented with 5 mM acetamide (only acetamidase-expressing cells can efficiently use acetamide as a nitrogen metabolite). To evaluate the yeast colonies, approximately 100–400 colonies may be taken from the pKS482 yeast plate. The inoculum from the colonies is cultured separately in 2.2 mL of K. lactis standard medium supplemented with 2% sugar alcohol as a carbon source. The cultures are incubated at 23.5°C for 6 days with shaking at 280 rpm, at which point the cell density in the culture reaches its maximum level, as indicated by the light absorbance at 600 nm (OD600). The cells are then removed from the culture by centrifugation at 4,000 rpm for 10 minutes, and the resulting supernatant (condition medium) is filtered through a 0.2 μM membrane for HPLC yield analysis.

[0360] Chemically synthesize peptides CRIP or peptide-IA can be produced by peptide synthesis, chemical synthesis, or using peptides and / or polypeptides. These methods can be carried out by those skilled in the art and / or through the use of commercial vendors (e.g., GenScript®; Piscataway, New Jersey). For example, in some embodiments, chemical peptide synthesis can be achieved using liquid-phase peptide synthesis (LPPS) or solid-phase peptide synthesis (SPPS).

[0361] In some embodiments, peptide synthesis can generally be achieved by using a strategy in which a nascent polypeptide chain is generated by coupling the carboxyl group of a subsequent amino acid to the N-terminus of a preceding amino acid (a process opposite to the type of polypeptide synthesis found in nature).

[0362] Peptide deprotection is a crucial first step in the chemical synthesis of polypeptides. Peptide deprotection is the process of blocking the reactive groups of amino acids by using chemicals to prevent them from participating in undesirable or nonspecific reactions or side reactions; in other words, the amino acids are "protected" from participating in these undesirable reactions.

[0363] Before synthesizing peptide chains, amino acids must be "deprotected" to allow chain formation (i.e., amino acids to bond). Chemicals used to protect the N-terminus include 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc), each of which can be removed by the use of a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoroacetic acid (TFA)).

[0364] The required C-terminal protector depends on the type of chemical peptide synthesis strategy used. For example, LPPS requires protection of the C-terminal amino acid, while SPPS does not require a solid support acting as a protecting group. Side-chain amino acids require the use of several different protecting groups, which vary depending on the individual peptide sequence and N-terminal protection strategy. However, typically, the protecting groups used for side-chain amino acids are based on tert-butyl (tBu) or benzyl (Bzl) protecting groups.

[0365] The next step in the peptide synthesis procedure is amino acid coupling. To achieve amino acid coupling, the C-terminal carboxylic acid of the incoming amino acid must be activated. This can be achieved using a carbodiimide such as diisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC), which reacts with the carboxyl group of the incoming amino acid to form an O-acylisourea intermediate. The O-acylisourea intermediate is then replaced by nucleophilic attack via the primary amino group at the N-terminus of the elongating peptide chain. The reactive intermediate produced by the carbodiimide can lead to amino acid racemization. To avoid amino acid racemization, a reagent such as 1-hydroxybenzotriazole (HOBt) is added to react with the O-acylisourea intermediate. Other coupling agents that may be used include 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), which have additional activating bases. Finally, deprotection and coupling of amino acids follow.

[0366] At the end of the synthesis process, the protecting groups from the polypeptide must be removed (usually through acid digestion). Determining which reagents are necessary for peptide cleavage depends on the protection scheme used and the overall synthesis method. For example, in some embodiments, hydrogen bromide (HBr), hydrogen fluoride (HF), or trifluoromethanesulfonic acid (TFMSA) can be used to cleave the Bzl and Boc groups. Alternatively, in other embodiments, a less strong acid such as TFA may be used to acid digest the tBut and Fmoc groups. Finally, the peptide can be purified based on its physiological and chemical characteristics (e.g., charge, size, hydrophobicity, etc.). Techniques that can be used to purify peptides include reversed-phase chromatography (RPC), size exclusion chromatography, partition chromatography, high-performance liquid chromatography (HPLC), and ion-exchange chromatography (IEC).

[0367] Exemplary methods of peptide synthesis are Anderson G.W. and McGregor A.C. (1957) T-butyloxycarbonylamino acids and their use in peptide synthesis. Journal of the American Chemical Society. 79, 6180-3, Carpino L.A. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1,1-disubstituted-2-arenesulfonhydrazides 1-4. Journal of the American Chemical Society. 79, 4427-31, McKay F.C. and Albertson N.F. (1957) New amine-masking groups for peptide synthesis. Journal of the American Chemical Society. 79, 4686-90, Merrifield R.B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 85, 2149-54, Carpino L.A. and Han G.Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry. 37, 3404-9, and A Lloyd-Williams P. et al. (1997) Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press.These can be found in U.S. Patent Nos. 278, 3,714,140 (filed March 16, 1971), 4,411,994 (filed June 8, 1978), 7,785,832 (filed January 20, 2006), 8,314,208 (filed February 10, 2006), and 10,442,834 (filed October 2, 2015), as well as U.S. Patent Application No. 2005 / 0165215 (filed December 23, 2004) (their disclosures are incorporated herein by reference in their entirety).

[0368] Further exemplary methods for generating polynucleotides, peptides, and CRIP can be found in U.S. Patent Application Publication No. 2015 / 0148288A1 (the disclosure thereof is incorporated herein by reference in its entirety).

[0369] Any of the methods described herein can be used to produce any of the CRIP, CRIP-insecticide protein, or peptide-IA described herein.

[0370] Cell culture technology and transformation technology The terms “transformation” and “transfection” both describe the process of introducing exogenous and / or heterologous DNA or RNA into a host organism. Generally, those skilled in the art may reserve the term “transformation” for the process of introducing exogenous and / or heterologous DNA or RNA into bacterial cells, and the term “transfection” for the process of introducing exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms “transformation” and “transfection” are used synonymously regardless of whether the process describes the introduction of exogenous and / or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plant, or animal).

[0371] In some embodiments, host cells may be transformed using the following methods: electroporation, cell compression, microinjection, impale infection, use of hydrostatic pressure, sonoporation, optical transfection, continuous injection, lipofection, use of viruses (e.g., adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus), chemical phosphate methods, endocytosis via DEAE-dextran or polyethyleneimine (PEI), protoplast fusion, hydrodynamic delivery, magnetofection, nucleoinfection, and / or other methods. Exemplary methods relating to transfection and / or transformation techniques can be found in Makrides (2003), Gene Transfer and Expression in Mammalian Cells, Elvesier; Wong, TK & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584-587 (1982); Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3; and Kim & Eberwine, Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010 Aug; 397(8): 3173-3178 (each of these references is incorporated herein by reference in whole).

[0372] Electroporation is a technique that applies electricity to cells to make the cell membrane permeable, thereby enabling the introduction of exogenous DNA into the cells. Electroporation is readily known to those skilled in the art, and the tools and devices necessary to achieve it are commercially available (e.g., Gene Pulser Xcell® electroporation system, Bio-Rad®, Neon® electroporation transfection system, Thermo-Fisher Scientific, and other tools and / or devices). Exemplary methods of electroporation are shown in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER:Unit-9.3, Saito (2015) Electroporation Methods in Neuroscience. Springer Press, and Pakhomov et al., (2017) Advanced Electroporation Techniques in Biology and Medicine. Taylor & Francis (these disclosures are incorporated herein by reference in their entirety).

[0373] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding CRIP or peptide-IA into yeast. For example, CRIP or peptide-IA can be cloned into a pKlac1 plasmid and transformed into K. lactis cells via electroporation. This is done by inoculating a suitable yeast species (e.g., Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc.) into about 10-200 mL of yeast extract peptone dextrose (YEPD) and culturing the yeast on a shaker at 30°C during the early logarithmic growth phase (e.g., about 0.6-2 × 10⁶). 8This can be achieved by incubation up to (cells / mL). The yeast is collected in a sterile centrifuge tube and centrifuged at 3000 rpm for 5 minutes at 4°C (Note: keep the cells cool during the procedure). The cells are washed with 40 mL of ice-cold sterile deionized water and pelleted at 23,000 rpm for 5 minutes. The washing step is repeated, and the cells are resuspended in 20 mL of 1 M fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol, hydrolyzed starch, isomalt, lactitol, maltitol, mannitol, and xylitol), followed by spinning down at 3,000 rpm for 5 minutes. The cells were resuspended in an appropriate amount of ice-cold 1M fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol, hydrolyzed starch, isomalt, lactitol, maltitol, mannitol, and xylitol) to obtain a final cell density of 3 × 10⁻⁶. 9 Adjust to cells / mL. Mix 40 μl of yeast suspension with approximately 1-4 μl of vector containing approximately 1 μg of linear polynucleotide encoding CRIP or peptide-IA in a pre-cooled 0.2 cm electroporation cuvette (Note: Ensure the sample is in contact with both sides of the aluminum cuvette). Provide a single pulse of 2000 V with an optimal time constant (5 ms) for the RC circuit, then recover the cells in a mixture of 0.5 mL of YED and 0.5 mL of 1 M fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol, hydrolyzed starch, isomalt, lactitol, maltitol, mannitol, and xylitol), then spread onto a selection plate.

[0374] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding CRIP or peptide-IA into a plant protoplast by: incubating sterile plant material in a plant protoplast solution (e.g., about 8 mL of 10 mM 2-[N-morpholino]ethanesulfonic acid (MES) (pH 5.5), 0.01% (w / v) pectilase, 1% (w / v) macerozyme, 40 mM CaCl2, and 0.4 M mannitol); adding the mixture to a rotary shaker at 30°C for about 3–6 hours to generate protoplasts; removing debris by filtration through an 80 μm mesh nylon screen; rinsing the screen with about 4 mL of plant electroporation buffer (e.g., 5 mM CaCl2, 0.4 M mannitol, and PBS). Combine the protoplasts in a sterile 15 mL conical centrifuge tube, then centrifuge at approximately 300 × g for approximately 5 minutes. After centrifugation, discard the supernatant and wash with 5 mL of plant electroporation buffer. Add the protoplasts to the plant electroporation buffer at a rate of approximately 1.5 × 10⁶ per 1 mL of liquid. 6 ~2×10 6 Resuspend the protoplasts in mL of protoplasts. Transfer approximately 0.5 mL of the protoplast suspension to one or more electroporation cubes, place on ice, and add the vector (Note: For stable transformation, the vector should be linearized using one of the restriction methods described above, and approximately 1–10 μg of vector should be used. For transient expression, the vector may be held in its supercoiled state, and approximately 10–40 μg of vector may be used). Mix the vector with the protoplast suspension. Place the cuvette in the electroporation apparatus and deliver one or more electric shocks at approximately 1–2 kV (a volume of 3–25 μF may be used initially while optimizing the reaction). Return the cuvette to ice and dilute the transformed cells 20-fold in complete medium. After approximately 48 hours, collect the protoplasts.

[0375] host cell The methods, compositions, CRIP, and peptide-IA of the present invention can be implemented in any cell type (e.g., eukaryotic or prokaryotic cells).

[0376] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA is a prokaryote. For example, in some embodiments, the host cell may be an archaeon or bacterium, such as a Gram-negative or Gram-positive organism. Examples of useful bacteria include the genera Escherichia coli (e.g., E. coli), Bacillus (e.g., B. subtilis), Enterobacteriaceae, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Sigella, Rhizobia, Vitreosilla, or Paracoccus.

[0377] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be a unicellular cell. For example, in some embodiments, the host cell may be a bacterial cell, such as a Gram-positive bacterium.

[0378] In some embodiments, the host cell can be a bacterium selected from the genus consisting of: Candidatus Chloracidobacterium, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mycobacterium, Propionibacterium, Streptomyces, Aquifex Bacteroides, Porphyromonas, Bacteroides, Porphyromonas, Flavobacterium, Chlamydia, Prosthecobacter, Verrucomicrobium, Chloroflexus, Chroococcus, Merismopedia, Synechococcus, Anabaena, Nostoc, Spirulina, Trichodesmium, Pleurocapsa, Prochlorococcus, Prochloron, Bacillus, Listeria, Staphylococcus, Clostridium, Dehalobacter, Epulopiscium, Ruminococcus, Enterococcus, Lactobacillus, Streptococcus, Erysipelothrix, Mycoplasma, Leptospirillum, Nitrospira, Thermodesulfobacterium, Gemmata, Pirellula, Planctomyces, Caulobacter, Agrobacterium, Bradyrhizobium, Brucella, Methylobacterium, Prosthecomicrobium, Rhizobium, Rhodopseudomonas, Sinorhizobium, Rhodobacter, Roseobacter, Acetobacter, Rhodospirillum, Rickettsia, Rickettsiaconorii, Mitochondria, Wolbachia, Erythrobacter, Erythromicrobium, Sphingomonas, Alcaligenes, Burkholderia, Leptothrix, Sphaerotilus, Thiobacillus, Neisseria, Nitrosomonas, Gallionella, Spirillum, Azoarcus, Aeromonas, Succinomonas, Succinivibrio, Ruminobacter, Nitrosococcus, Thiocapsa, Enterobacter, Escherichia, Klebsiella, Salmonella, Shigella, Wigglesworthia, Yersinia, Coxiella, Legionella, Halomonas, Pasteurella, Acinetobacter, Azotobacter, Pseudomonas, Psychrobacter, Beggiatoa, Thiomargarita, Vibrio, Xanthomonas, Bdellovibrio, Campylobacter, Helicobacter, Myxococcus, Desulfosarcina, Geobacter, Desulfuromonas, Borrelia, Leptospira, Treponema, Petrotoga, Thermotoga, Deinococcus, or Thermus.

[0379] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be selected from one of the following bacterial species: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptomyces lividans, Streptomyces murinus, Streptomyces coelicolor, Streptomyces albicans, Streptomyces griseus, Streptomyces plicatosporus, Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia senegalensis, Escherichia vulneris, Pseudomonas abietaniphila, Pseudomonas agarici, Pseudomonas agarolyticus, Pseudomonas alcaliphila, Pseudomonas alginovora, Pseudomonas andersonii, Pseudomonas antarctica, Pseudomonas asplenii, Pseudomonas azelaica, Pseudomonas batumici, Pseudomonas borealis, Pseudomonas brassicacearum, Pseudomonas chloritidismutans, Pseudomonas cremoricolorata, Pseudomonas diterpeniphila, Pseudomonasfiliscindens, Pseudomonas frederiksbergensis, Pseudomonas gingeri, Pseudomonas graminis, Pseudomonas grimontii, Pseudomonas halodenitrificans, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas hydrogenovora, Pseudomonas indica, Pseudomonas japonica, Pseudomonas jessenii, Pseudomonas kilonensis, Pseudomonas koreensis, Pseudomonas lini, Pseudomonas lurida, Pseudomonas lutea, Pseudomonas marginata, Pseudomonas meridiana, Pseudomonas mesoacidophila, Pseudomonas pachastrellae, Pseudomonas palleroniana, Pseudomonas parafulva, Pseudomonas pavonanceae, Pseudomonas proteolyica, Pseudomonas psychrophila, Pseudomonas psychrotolerans, Pseudomonas pudica, Pseudomonas rathonis, Pseudomonas reactans, Pseudomonas rhizosphaerae, Pseudomonas salmononii、Pseudomonas thermaerum、Pseudomonas thermocarboxydovorans、Pseudomonas thermotolerans、Pseudomonas thivervalensis、Pseudomonas umsongensis、Pseudomonas vancouverensis、Pseudomonas wisconsinensis、Pseudomonas xanthomarina Pseudomonas xiamenensis、Pseudomonas aeruginosa、Pseudomonasalcaligenes, Pseudomonas anguilliseptica, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas jinjuensis, Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminae, Pseudomonas aurantiaca, Pseudomonas chlororaphis, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas taetrolens Pseudomonas azotoformans, Pseudomonas brenneri, Pseudomonas cedrina, Pseudomonas congelans, Pseudomonas corrugata, Pseudomonas costantinii, Pseudomonas extremorientalis, Pseudomonas fluorescens, Pseudomonas fulgida, Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas poae, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas tolaasii, Pseudomonas trivialis, Pseudomonas veronii Pseudomonas denitrificans, Pseudomonas pertucinogena, Pseudomonas fulva, Pseudomonas monteilii, Pseudomonasmosselii, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas balearica, Pseudomonas luteola, or Pseudomonas stutzeri. Pseudomonas avellanae, Pseudomonas cannabina, Pseudomonas caricapapyae, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas fuscovaginae, Pseudomonas tremae, or Pseudomonas viridiflava.

[0380] In some embodiments, the host cell used to produce CRIP, CRIP-insecticide protein, or peptide-IA may be a eukaryote.

[0381] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be cells belonging to the following clades: Opisthokonta, green plants (e.g., algae and plants), Amoebozoa, Cercozoa, Alveolata, Flagellates, Heterokonta, Disicristata, or Excavata.

[0382] In some embodiments, the procedures and methods described herein can be achieved using host cells, for example, metazoans, choanoflagellates, or fungi.

[0383] In some embodiments, the procedures and methods described herein can be achieved using a host cell that is a fungus. For example, in some embodiments, the host cell may be a cell belonging to one of the following eukaryotes: Ascomycota, Basidiomycota, Chytridiomycota, Microsporidia, or Zygomycota.

[0384] In some embodiments, the procedures and methods described herein can be achieved using a host cell that is a fungus belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.

[0385] In some embodiments, the procedures and methods described herein can be achieved using a host cell that is a fungus belonging to one of the following species: Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces uvarum, Aspergillus flavus, A. terreus, A. awamori, Cladosporium elatum, Cl. Herbarum, Cl. Sphaerospermum, and Cl. Cladosporioides, Magnaporthe grise, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0386] In some embodiments, the procedures and methods described herein can be achieved using host cells that are Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, or Pichia pastoris.

[0387] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.

[0388] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be a member of the Saccharomycetaceae family. For example, in some embodiments, the host cell may be one of the following genera within the Saccharomycetaceae family: Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.

[0389] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[0390] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be species within the genus Candida. For example, the host cell can be one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[0391] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be a species within the genus Kluyveromyces. For example, the host cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.

[0392] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be species within the genus Pichia. For example, the host cells may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.

[0393] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be species within the genus Saccharomyces. For example, the host cell can be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[0394] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be one of the following: Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, or Hansenula anomala.

[0395] The use of yeast cells as a host organism for generating recombinant CRIP or peptide-IA is an exceptional method well known to those skilled in the art. In some embodiments, the methods and compositions described herein can be carried out using any species of yeast (including, but not limited to, any species of the genus Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces), where Saccharomyces species include any species of Saccharomyces, for example, the Saccharomyces cerevisiae species is selected from the following strains: INVSc1, YNN27, S150-2B, W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A, and BJ3505. In some embodiments, members of the genus Pichia include any species of the genus Pichia (e.g., Pichia pastoris), for example, Pichia pastoris is selected from the following strains: Bg08, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, KM71, MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any pep4 knockout strain and any prb1 knockout strain, as well as Pichia selected from the following strains Pastoris: Bg08, X-33, SMD1168, and KM71.In some embodiments, the methods described herein can be achieved using any species of the genus Kluyveromyces, including any species of the genus Kluyveromyces (e.g., Kluyveromyces lactis). As taught by the inventors, the staining of Kluyveromyces lactis may be from the following strains, but selection is not necessarily required: GG799, YCT306, YCT284, YCT389, YCT390, YCT569, YCT598, NRRL Y-1140, MW98-8C, MS1, CBS293.91, Y721, MD2 / 1, PM6-7A, WM37, K6, K7, 22AR1, 22A295-1, SD11, MG1 / 2, MSK110, JA6, CMK5, HP101, HP108, and PM6-3C. The lactis species are selected from GG799, YCT306, and NRRL Y-1140.

[0396] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Aspergillus oryzae.

[0397] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Aspergillus japonicas.

[0398] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Aspergillus niger.

[0399] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Bacillus licheniformis.

[0400] In some embodiments, the host cells used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Bacillus subtilis.

[0401] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Trichoderma reesei.

[0402] In some embodiments, the procedures and methods described herein can be achieved using a host cell that is yeast. The yeast includes, but is not limited to, any species of Hansenula, and preferably Hansenula polymorpha. In some embodiments, the procedures and methods described herein can be achieved with any species of yeast, including, but is not limited to, any species of Yarrowia, such as Yarrowia lipolytica. In some embodiments, the procedures and methods described herein can be achieved with any species of yeast, including, but is not limited to, any species of Schizosaccharomyces, and preferably Schizosaccharomyces pombe.

[0403] yeast cell culture In some embodiments, yeast species, such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as host organisms. Yeast cell culture techniques are well known to those skilled in the art. Exemplary methods for yeast cell culture include Evans, Yeast Protocols. Springer (1996), Bill, Recombinant Protein Production in Yeast. Springer (2012), Hagan et al., Fission Yeast: A Laboratory Manual. CSH Press (2016), Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture. Biosci Biotechnol Biochem. 2014;78(6):1090-3, Dymond, Saccharomyces cerevisiae growth media. Methods Enzymol. 2013;533:191-204, Luke et al., Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 2011 May;50(5):325-8, and Romanos et al., Culture of yeast for the production of heterologous proteins. Curr Protoc Cell Biol. 2014 Sep 2;64:20.9.1-16 (the disclosure thereof is incorporated herein by reference in its entirety).

[0404] The recipes for yeast cell fermentation media and stocks are as follows: (1) MSM medium recipe: 2 g / L sodium citrate dihydrate, 1 g / L calcium sulfate dihydrate (0.79 g / L anhydrous calcium sulfate), 42.9 g / L monopotassium phosphate, 5.17 g / L ammonium sulfate, 14.33 g / L potassium sulfate, 11.7 g / L magnesium sulfate heptahydrate, 2 mL / L PTM1 trace salt solution, 0.4 ppm biotin (500 ×, from 200 ppm stock), 1-2% pure glycerol or other carbon source. (2) PTM1 trace salt solution: 6.0g copper sulfate-5H2O, 0.08g sodium iodide, 3.0g manganese sulfate-H2O, 0.2g sodium molybdate-2H2O, 0.02g boric acid, 0.5g cobalt chloride, 20.0g zinc chloride, 65.0g iron sulfate-7H2O, 0.2g biotin, 5.0ml sulfuric acid, and water are added to make a final volume of 1 liter. An exemplary composition of K. lactis standard medium (DMSor) is as follows: 11.83 g / L KH2PO4, 2.299 g / L K2HPO4, 20 g / L fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol, hydrolyzed starch, isomalt, lactitol, maltitol, mannitol, and xylitol), 1 g / L MgSO4.7H2O, 10 g / L (NH4)SO4, 0.33 g / L CaCl 2. 2H2O, 1g / L; NaCl, 1g / L; KCl, 5mg / L; CuSO4.5H2O, 30mg / L; MnSO4.H2O, 10mg / L; ZnCl2, 1mg / L; KI, 2mg / L; CoCl2.6H2O, 8mg / L; Na2MoO4.2H2O, 0.4mg / L; H3BO3, 15mg / L; FeCl3.6H2O, 0.8mg / L; Biotin, 20mg / L; Calcium pantothenate, 15mg / L; Thiamine, 16mg / L; Myo-inositol, 10mg / L; Nicotinic acid, and 4mg / L; Pyridoxine.

[0405] Yeast cells can be cultured in 48-well deep-well plates (sealed with a sterile, airtight cover after inoculation). Colonies of yeast (e.g., K. lactis) cultured on the plate can be taken and inoculated into a deep-well plate containing 2.2 mL of medium per well (consisting of DMSor). The inoculated deep-well plate can be grown at 23.5°C for 6 days with shaking at 280 rpm in a refrigerated incubator shaker. On day 6 after inoculation, the medium should be collected by centrifugation at 4000 rpm for 10 minutes. Subsequently, it is filtered using a filtration plate with a 0.22 μM membrane, and the filtered medium is subjected to HPLC analysis.

[0406] Yeast transformation, peptide purification, and analysis An exemplary method of yeast transformation is as follows: Yeast cells are transformed with an expression vector carrying a CRIP ORF, a CRIP-insecticide protein ORF, or a peptide-IA ORF. First, the expression vector is linearized by specific restriction enzyme digestion, usually to facilitate integration into the chromosome via homologous recombination. The linear expression vector is then transformed into yeast cells by chemical transformation methods or electroporation, and integrated into target loci of the yeast genome by homologous recombination. Integration may occur multiple times at the same chromosomal locus. Thus, the genome of the transformed yeast cells may contain multiple copies of the CRIP or peptide-IA expression cassette. Successfully transformed yeast cells can be engineered with the expression vector and identified using growth conditions that favor selective markers in which CRIP, CRIP-insecticide protein, or peptide-IA ORF are integrated together into the yeast chromosome. Examples of such markers, but not limited to, include acetamide prototrophy, zeosin resistance, genetisin resistance, knowureotricin resistance, and uracil prototrophy.

[0407] Due to the influence of unpredictable and variable factors (e.g., epigenetic modifications of genes and gene networks, and variations in the number of integration events occurring in individual cells of a population undergoing the transformation procedure), individual yeast colonies undergoing a given transformation process will have different abilities to produce CRIP ORFs, CRIP-insecticidal protein ORFs, or peptide-IA ORFs. Therefore, transgenic yeast colonies carrying CRIP or peptide-IA transgenes should be screened for high-yield strains. Two effective methods for such screening (each depending on the growth of small-scale cultures of transgenic yeast to provide condition medium samples for subsequent analysis) involve analyzing condition medium samples from positive transgenic yeast colonies using reverse-phase HPLC or a housefly injection procedure.

[0408] Transgenic yeast culture can be performed using 14 mL round-bottom polypropylene culture tubes with 5–10 mL of standard medium added to each tube, or in a 48-well deep-well culture plate with 2.2 mL of standard medium added to each well. Standard medium free of crude protein extracts or by-products (e.g., yeast extract or peptone) is used for culture to reduce the protein background in the condition medium recovered for the subsequent screening step. Culture is carried out at an optimal temperature (e.g., 23.5°C for K. lactis) for approximately 5–6 days until the maximum cell density is reached. At this point, CRIP or peptide-IA will be produced by the transformed yeast cells and secreted from the cells into the growth medium. To prepare samples for screening, cells are removed from the culture by centrifugation, the supernatant is recovered as condition medium, and then purified by filtration through a 0.22 μm filter membrane, and then prepared for strain screening.

[0409] In some embodiments, positive yeast colonies transformed with CRIP or peptide-IA can be screened via reverse-phase HPLC (rpHPLC) screening of putative yeast colonies. This screening method can use an HPLC analytical column with a C18 bound phase. Acetonitrile and water are used as the mobile phase solvent, and an ultraviolet absorbance detector set to 220 nm is used for peptide detection. An appropriate amount of conditional medium sample is loaded into the rpHPLC system and eluted with a linear gradient of the mobile phase solvent. The concentration of CRIP or peptide-IA in the conditional medium is quantified using the corresponding peak area of ​​the insecticidal peptide in the HPLC chromatograph. A known amount of pure CRIP or peptide-IA is run through the same rpHPLC column using the same HPLC protocol to confirm the retention time of the peptide and generate a standard peptide HPLC curve for quantification.

[0410] An exemplary reverse-phase HPLC screening process for positive K. lactis cells is as follows: A CRIP ORF, CRIP-insecticidal protein ORF, or peptide-IA ORF can be inserted into the expression vector pKLAC1 and transformed into K. lactis strain YCT306 (from New England Biolabs, Ipswich, MA, USA). The pKLAC1 vector is an embedded expression vector. Once the CRIP or peptide-IA transgene was cloned into pKLAC1 and transformed into YCT306, their expression was controlled by the LAC4 promoter. The resulting transformed colonies produced a prepropeptide containing an α-conjugation factor signal peptide, a Kex2 cleavage site, and mature CRIP or peptide-IA. The α-conjugation factor signal peptide induced the prepropeptide to enter the endogenous secretory pathway, releasing mature CRIP or peptide-IA into the growth medium.

[0411] In some embodiments, codon optimization for CRIP or peptide-IA expression may be performed in two rounds. For example, in the first round, an expressing α-junction signal peptide, a Kex2 cleavage site, and CRIP or peptide-IA are designed based on several common features of multiple variants of a high-expression DNA sequence and a CRIP or peptide-IA expression ORF, and their expression levels are evaluated in the K. lactis YCT306 strain to yield an initial K. lactis expression algorithm. In the second round of optimization, additional variant CRIP or peptide-IA expression ORFs are designed based on the initial K. lactis expression algorithm to further refine the K. lactis expression algorithm and identify the best ORF for CRIP or peptide-IA expression in K. lactis. In some embodiments, the DNA sequence obtained from the above optimization may have an α-MF signal peptide, a Kex2 cleavage site, and an open reading frame encoding CRIP, the CRIP-insecticidal protein, or peptide-IA. These can be cloned into a pKLAC1 vector using the Hind III and Not I restriction sites to obtain a CRIP or peptide-IA expression vector.

[0412] In some embodiments, the yeast Pichia pastoris can be transformed with CRIP, CRIP-insecticidal protein, or peptide-IA expression cassettes. An exemplary method for transforming P. pastoris is as follows: P. pastoris can be transformed with CRIP or peptide-IA using the vectors pJUGαKR and pJUZαKR. The pJUGαKR and pJUZαKR vectors are available from Biogrammatics, Carlsbad, California, USA. Both vectors are embedded vectors and use a uracil phosphoribosyltransferase promoter (pUPP) to enhance the expression of xenotransgenes. The only difference between the vectors is that pJUGαKR provides G418 resistance to the host yeast, while pJUZαKR provides zeosin resistance. Pairs of complementary oligonucleotides encoding CRIP or peptide-IA are designed and synthesized for subcloning into the two yeast expression vectors. The hybridization reaction involves mixing the corresponding complementary oligonucleotides to a final concentration of 20 μM in 30 mM NaCl and 10 mM Tris-Cl (pH 8) (all final concentrations), then incubating at 95°C for 20 minutes, followed by incubation for 9 hours, starting at 92°C and ending at 17°C, with the temperature decreasing by 3°C every 20 minutes. The hybridization reaction yields DNA fragments encoding CRIP or peptide-IA. Two P. pastoris vectors are digested with BsaI-HF restriction enzyme, and the double-stranded DNA product of this reaction is subcloned into a linearized P. pastoris vector using standard procedures. After validation of the subclone sequences, aliquots of the plasmid are transfected into P. pastoris strain Bg08 by electroporation. The resulting transformed yeasts can be selected based on their resistance to zeosin or G418, which is conferred to the vectors pJUZαKR and pJUGαKR, respectively, by the manipulated elements, and can be cultured and screened as described herein.

[0413] A detailed description of ORF and its components is provided below.

[0414] Screening and evaluation of yeast peptide yield Peptide yield can be determined by any method known to those skilled in the art (e.g., capillary gel electrophoresis (CGE), Western blot analysis, etc.). Activity assays, as described herein and known in the art, can also provide information regarding peptide yield. In some embodiments, peptide yield can be evaluated using these or any other method known in the art.

[0415] Quantitative assay In some embodiments, but not limited to, CRIP peptide yield can be measured using: HPLC, mass spectrometry (MS) and related techniques, LC / MS / MS, reverse-phase protein array (RPPA), immunohistochemistry, ELISA, suspension bead array, mass spectrometry, dot blotting, SDS-PAGE, capillary gel electrophoresis (CGE), Western blotting, Bradford assay, measurement of UV absorption at 260 nm, Lowry assay, Smith copper / bicinchoninate assay, secretion assay, Pierce protein assay, Biuret reaction, etc. Exemplary methods for protein quantification include Stoscheck, C. 1990 “Quantification of Protein” Methods in Enzymology, 182:50-68; Lowry, O. Rosebrough, A., Farr, A. and Randall, R. 1951 J. Biol. Chem. 193:265; Smith, P. et al., (1985) Anal. Biochem. 150:76-85; Bradford, M. 1976 “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding” Anal. Biochem. 72:248-254; Cabib, E. and Polacheck, I. 1984 “Protein assay for dilute solutions.” Methods in Enzymology, 104:318-328; Turcanu, Victor; Williams, Neil A. (2001). "Cell identification and isolation on the basis of cytokine secretion: A novel tool for investigating immune responses." Nature Medicine. 7(3):373-376; provided in U.S. Patent No. 6,391,649 (their disclosures are incorporated herein by reference in their entirety).

[0416] In other embodiments, the CRIP peptide yield may be quantified and / or evaluated using methods including, but not limited to, the following: the amount of recombinant protein per volume of culture (e.g., grams or milligrams of protein per liter of culture); the percentage or fraction of recombinant protein insoluble precipitate obtained after cell lysis (e.g., the amount of recombinant protein extracted from the supernatant / the amount of protein in the insoluble component); the percentage or fraction of active protein (e.g., the amount of active protein for use in the protein amount / analysis); the percentage or fraction of total cellular protein (tcp); and / or the amount of protein / the percentage or ratio of cells and dry biomass.

[0417] In some embodiments, yield is expressed in relation to culture volume, and in particular, when comparing yields between different cultures, cell density may be taken into consideration.

[0418] In some embodiments, the present invention provides a method for producing heterologous polypeptides in an amount of at least about 5%, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or more of total cellular protein (tcp). "Percentage of total cellular protein (%)" is the amount of heterologous polypeptide in the host cell as a percentage of aggregated cellular protein. The determination of the percentage of total cellular protein is well known in the art.

[0419] "Total cellular protein (tcp)" or "percentage of total cellular protein (% tcp)" is the amount of protein or polypeptide in a host cell as a percentage of aggregated cellular protein. Methods for determining the percentage of total cellular protein are well known in the art.

[0420] In some embodiments, peptide yield can be quantified using HPLC. For example, in some embodiments, the yield of CRIP or peptide-IA can be evaluated using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 × 100 mm C18 reversed-phase HPLC column and an auto-injector. An exemplary use of the Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 × 100 mm C18 reversed-phase HPLC column and an auto-injector is as follows: A filtered conditional medium sample derived from transformed K. lactis cells is analyzed using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 × 100 mm C18 reversed-phase HPLC column and an auto-injector by analyzing HPLC-grade water and acetonitrile containing 0.1% trifluoroacetic acid, which constitute the two mobile phase solvents used for HPLC analysis. The peak areas of both CRIP and peptide-IA can be analyzed using HPLC chromatography and then used to calculate the peptide concentration in the conditional medium, which can be further normalized to the corresponding final cell density (determined by OD600 measurement) as a normalized peptide yield.

[0421] Activity assay In some embodiments, positive yeast colonies transformed with CRIP or peptide-IA can be screened using a housefly injection assay. CRIP or peptide-IA can paralyze / kill houseflies when injected through the body wall of the dorsal thorax at the measured dose. The efficacy of CRIP or peptide-IA is measured by the median paralyzing / lethal dose (PD) of the peptide. 50 / LD 50 ) can be defined by this, which results in a 50% knockdown rate or mortality rate in injected houseflies, respectively. Pure CRIP or peptide-IA is typically used in housefly injection assays and PD 50 / LD 50 Generate a standard dose-response curve from which values ​​can be determined. PD from analysis of standard dose-response curves of pure CRIP or peptide-IA.50 / LD 50 Using the values, quantification of CRIP or peptide-IA produced by transformed yeast can be achieved using a housefly injection assay (performed using serial dilutions of the corresponding conditional medium).

[0422] An exemplary housefly injection bioassay is as follows: The condition medium is serially diluted to generate a complete dose-response curve from the housefly injection bioassay. Before injection, adult houseflies (Musca domestica) are immobilized with CO2, and 12–18 mg of houseflies are selected for injection. Using a microapplicator loaded with a 1 cc syringe and a 30 gauge needle, 0.5 μL of serially diluted condition medium sample per fly is injected into the houseflies through the body wall of the dorsal thorax. The injected houseflies are placed in a sealed container with moistened filter paper on the lid and a breathing hole, and they are examined 24 hours after injection by scoring the knockdown rate or mortality rate. The normalized yield is calculated. Peptide yield refers to the peptide concentration in mg / L units in the condition medium. However, peptide yield is not always sufficient to accurately compare the production rates of strains. Individual strains may have different growth rates. Therefore, when the cultures are harvested, different cultures may have different cell densities. Even if a strain has a lower peptide production rate than another strain with a higher production rate, a culture with a high cell density may produce a higher concentration of peptide in the medium. Therefore, the term "normalized yield" is created by dividing the peptide yield by the cell density of the corresponding culture, which allows for a better comparison of peptide production rates between strains. Cell density is expressed in units of "A" (absorbance units) by the absorbance of light at 600 nm.

[0423] By screening yeast colonies transformed with CRIP or peptide-IA, high-yield yeast strains can be identified from hundreds of potential colonies. When these strains are fermented in a bioreactor using the optimized fermentation media and conditions described herein, yields of at least 4 g / L, or at least 3 g / L, or at least 2 g / L of CRIP or peptide-IA can be achieved. Higher production rates (expressed in mg / L) are approximately 100 mg / L to 100,000 mg / L, or approximately 100 mg / L to 90,000 mg / L, or approximately 100 mg / L to 80,000 mg / L, or approximately 100 mg / L to 70,000 mg / L, or approximately 100 mg / L to 60,000 mg / L, or approximately 100 mg / L to 50,000 mg / L, or approximately 100 mg / L to 40,000 mg / L, or approximately 100 mg / L to approximately 30,000 mg / L, or approximately 100 mg / L to approximately 20,000 mg / L, or approximately 100 mg / L to approximately 17,500 mg / L, or approximately 100 mg / L to approximately 15,000 mg / L, or approximately 100 mg / L to approximately 12,500 mg / L, or approximately 100 mg / L to approximately 10,000 mg / L, or approximately 100 mg / L to approximately 9,000 mg / L, or approximately 100 mg / L to approximately 8,000 mg / L, or approximately 100 mg / L to approximately 7,000 mg / L, or approximately 100 mg / L to approximately 6,000 mg / L, or approximately 100 mg / L to approximately 5,000 mg / L, or approximately 100 mg / L to approximately 3,000 mg / L, or approximately 100 mg / L to approximately 2,000 mg / L, or approximately 100 mg / L to 1,500 mg / L, or approximately 100 mg / L to 1,000 mg / L, or approximately 100 mg / L to 750 mg / L, or approximately 100 mg / L to 500 mg / L, or approximately 150 mg g / L to 100,000 mg / L, or approximately 200 mg / L to 100,000 mg / L, or approximately 300 mg / L to 100,000 mg / L, or approximately 400 mg / L to 100,000 mg / L, or approximately 500 mg / L to 100,000 mg / L, or approximately 750 mg / L to 100,000 mg / L, or approximately 1,000 mg / L to 100,000 mg / L, or approximately 1,250 mg / L to 100,000 mg / L, or approximately 1,500 mg / L to 100,000 mg / L, or approximately 2,000 mg / L to 100,000 mg / L, or approximately 2,500 mg / L to 100,000 mg / L, or approximately 3,000 mg / L to 100,000 mg / L, or approximately 3,500 mg / L to 100,000 mg / L, or approximately 4,000 mg / L to 100,000 mg / L, or approximately 4,500 mg / L to 100,000 mg / L, or approximately 5,000 mg / L to 100,000 mg / L, or approximately 6,000 mg / L to 100,000 mg / L, or approximately 7,000 mg / L to 100,000 mg / L, or approximately 8,000 mg / L to 100,000 mg / L, or approximately 9,000 mg / L to 100,000 mg / L, or approximately 10,000 mg / L to 100,000 mg / L, or approximately 12,500 mg / L to 100,000 mg / L, Or approximately 15,000 mg / L to 100,000 mg / L, or approximately 17,500 mg / L to 100,000 mg / L, or approximately 20,000 mg / L to 100,000 mg / L, or approximately 30,000 mg / L to 100,000 mg / L, or approximately 40,000 mg / L to 100,000 mg / L, or approximately 50,000 mg / L to 100,000 mg / L, or approximately 60,000 mg / L to 100, The yield may be 000 mg / L, or approximately 70,000 mg / L to 100,000 mg / L, or approximately 80,000 mg / L to 100,000 mg / L, or approximately 90,000 mg / L to 100,000 mg / L, or any range of any of the values ​​provided, or a yield even higher than the yield that can be achieved with the pre-conversion peptide using the same or similar production method used to produce the peptide before conversion.

[0424] CRIP and / or peptide-IA (e.g., insecticides subjected to such methods, e.g., polymers of amino acids, peptides, and / or proteins) can be produced by using and / or modifying any of the methods described above. For example, using any of the methods described above, any of the CRIP or peptide-IA described herein, including but not limited to ACTX peptides (e.g., U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, rκ-ACTX-Hv1c, ω-ACTX-Hv1a, and / or ω-ACTX-Hv1a+2), Γ-CNTX-Pn1a, U1-agatoxin-Ta1b, TVP, Av2, Av3, AVP, and / or Bt toxins (e.g., Cry toxin, Cyt toxin, or Vip), can be produced, generated, prepared, expressed, transcribed, translated, synthesized, or otherwise prepared.

[0425] Culture conditions and fermentation conditions Cell culture techniques are well known in the art. In some embodiments, the culture method and / or materials will invariably require adaptation based on selected host cells, and such adaptations (e.g., modifying pH, temperature, culture medium composition, etc.) are well known to those skilled in the art. In some embodiments, CRIP, CRIP-insecticidal protein, or peptide-IA of the present invention can be produced using any known culture technique.

[0426] Exemplary cultivation methods are provided in U.S. Patents 3,933,590, 3,946,780, 4,988,623, 5,153,131, 5,153,133, 5,155,034, 5,316,905, 5,330,908, 6,159,724, 7,419,801, 9,320,816, 9,714,408, and 10,563,169 (their disclosures are incorporated herein by reference in their entirety).

[0427] yeast culture Yeast cell culture techniques are well known to those skilled in the art. Exemplary methods for yeast cell culture include Evans, Yeast Protocols. Springer (1996), Bill, Recombinant Protein Production in Yeast. Springer (2012), Hagan et al., Fission Yeast: A Laboratory Manual. CSH Press (2016), Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in pre-culture. Biosci Biotechnol Biochem. 2014;78(6):1090-3, Dymond, Saccharomyces cerevisiae growth media. Methods Enzymol. 2013;533:191-204, Luke et al., Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 2011 May;50(5):325-8, and Romanos et al., Culture of yeast for the production of heterologous proteins. Curr Protoc Cell Biol. 2014 Sep 2;64:20.9.1-16 (the disclosure thereof is incorporated herein by reference in its entirety).

[0428] Yeast can be cultured in a variety of media. For example, in some embodiments, yeast can be cultured in minimal medium, YPD medium, yeast synthesis dropout medium, yeast nitrogen source basal medium (YNB, with or without amino acids), YEPD medium, ADE D medium, ADE DS medium, LEU D medium, HIS D medium, or mineral salt medium.

[0429] In some embodiments, yeast can be cultured in minimal medium. In some embodiments, the minimal medium components may include 2% sugar, phosphate buffer (pH 6.0), magnesium sulfate, calcium chloride, ammonium sulfate, sodium chloride, potassium chloride, copper sulfate, manganese sulfate, zinc chloride, potassium iodide, cobalt chloride, sodium molybdate, boric acid, iron chloride, biotin, calcium pantothenate, thiamine, myo-inositol, nicotinic acid, and pyridoxine.

[0430] In some embodiments, yeast can be cultured in YPD medium, which comprises bacteriological peptone, yeast extract, and glucose.

[0431] In some embodiments, yeast can be cultured in a yeast synthetic dropout medium, which can be used to distinguish between nutrient-requiring mutants that cannot grow without certain culture medium components and are transformed with plasmids that allow the transformants to grow in media lacking necessary components.

[0432] In some embodiments, yeast can be cultured using a yeast nitrogen source basal medium (YNB) containing or not containing amino acids, which contains nitrogen, vitamins, trace elements, and salts.

[0433] In some embodiments, the culture medium may be YEPD medium, for example, a medium containing 2% D-glucose, 2% bactopeptone (Difco Laboratories, Detroit, MI), 1% bacto yeast extract (Difco), 0.004% adenine, and 0.006% L-leucine, or a variation thereof, and the carbon source is a sugar alcohol (e.g., glycerol or sorbitol).

[0434] In some embodiments, the culture medium may be ADE D medium, for example, a medium containing 0.056% Ade-Trp-Thr powder, a 0.67% yeast nitrogen source basic medium without amino acids, 2% D-glucose, and a 0.5% 200× tryptophan threonine solution, or a variation thereof, wherein the carbon source is a sugar alcohol (e.g., glycerol or sorbitol).

[0435] In some embodiments, the culture medium may be ADE DS" medium, for example, a medium containing 0.056% Ade-Trp-Thr powder, a 0.67% yeast nitrogen source basic medium without amino acids, 2% D-glucose, 0.5% 200× tryptophan threonine solution, and 18.22% D-sorbitol, or a variation thereof, wherein the carbon source is entirely a sugar alcohol (e.g., glycerol or sorbitol).

[0436] In some embodiments, the culture medium may be LEU D medium, for example, a medium containing 0.052% Leu-Trp-Thr powder, a 0.67% yeast nitrogen source basic medium without amino acids, 2% D-glucose, and a 0.5% 200× tryptophan threonine solution, or a variation thereof, wherein the carbon source is a sugar alcohol (e.g., glycerol or sorbitol).

[0437] In some embodiments, the culture medium may be HIS D medium, for example, a medium containing 0.052%-His-Trp-Thr powder, 0.67% yeast nitrogen source basic medium without amino acids, 2% D-glucose, and 0.5% 200×tryptophanthreonine solution, or a variation thereof, and the carbon source is a sugar alcohol (e.g., glycerol or sorbitol).

[0438] In some embodiments, mineral salt media can be used. Mineral salt media consist of mineral salts and a carbon source (e.g., glucose, sucrose, or glycerol). Examples of mineral salt media include, for example, M9 medium, Pseudomonas medium (ATCC 179), and Davis-Mingioli medium. See Davis & Mingioli (1950) J.Bact. 60:17-28. Mineral salts used to prepare mineral salt media include, for example, potassium phosphate, ammonium sulfate or ammonium chloride, magnesium sulfate or magnesium chloride, and trace minerals (e.g., calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc). Typically, organic nitrogen sources such as peptone, tryptone, amino acids, or yeast extracts are not included in mineral salt media. Instead, inorganic nitrogen sources are used, which may be selected from, for example, ammonium salts, aqueous ammonia solutions, and gaseous ammonia. Mineral salt media typically contain glucose or glycerol as the carbon source.

[0439] Compared to mineral salt media, minimal media may also contain mineral salts and a carbon source, but may be supplemented with, for example, low levels of amino acids, vitamins, peptones, or other components (however these are added in very minimal levels). The media can be prepared using, for example, the method described in U.S. Patent Application Publication No. 2006 / 0040352 (the disclosure thereof is incorporated herein by reference in its entirety). Details of the culture procedure and mineral salt media useful for the method of the present invention are described in Riesenberg, D et al., 1991, “High cell density cultivation of Escherichia coli at controlled specific growth rate,” J. Biotechnol. 20(1):17-27.

[0440] In some embodiments, Kluyveromyces lactis is grown in minimal medium supplemented with 2% glucose, galactose, sorbitol, or glycerol as the sole carbon source. The cultures are incubated at 30°C for logarithmic metaphase (24–48 hours) for β-galactosidase measurement, or at 23.5°C for 6 days for heterologous protein expression.

[0441] In some embodiments, yeast cells can be cultured in a 48-well deep-well plate (sealed with a sterile, airtight cover after inoculation). Colonies of yeast (e.g., K. lactis) cultured on the plate can be taken and inoculated into a deep-well plate containing 2.2 mL of medium per well (consisting of DMSor). The inoculated deep-well plate can be grown at 23.5°C for 6 days with shaking at 280 rpm in a refrigerated incubator shaker. On day 6 after inoculation, the medium should be collected by centrifugation at 4000 rpm for 10 minutes. Subsequently, it is filtered using a filtration plate with a 0.22 μM membrane, and the filtered medium is subjected to HPLC analysis.

[0442] In some embodiments, yeast species (e.g., Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others) can be used as host organisms and / or yeast modified using the methods described herein.

[0443] Temperature and pH conditions vary depending on the stage of culture and the selected host cell type. Variables such as temperature and pH in cell culture are readily known to those skilled in the art.

[0444] pH level is important in yeast culture. Those skilled in the art will understand that the culture process includes not only initiating the yeast culture but also maintaining it. While yeast culture can be started at any pH level, care must be taken to monitor the pH level during the culture process, as the culture medium tends to become more acidic (i.e., the pH decreases) over time.

[0445] In some embodiments of the present invention, yeast is grown in a medium with a pH level required based on the yeast species used, the stage of culture, and / or temperature. Therefore, in some embodiments, the pH level can be in the range of about 2 to about 10. Those skilled in the art will recognize that the optimal pH for most microorganisms is around the neutral point (pH 7.0). However, in some embodiments, some fungal species prefer an acidic environment. Therefore, in some embodiments, the pH can be in the range of 2 to 6.5. In some embodiments, the pH can be in the range of about 4 to about 4.5. Some fungal species (e.g., molds) can grow at a pH of about 2 to about 8.5, but prefer an acidic pH. See Mountney & Gould, Practical food microbiology and technology. 1988. Ed. 3, and Pena et al., Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae. FEMS Yeast Res. 2015 Mar;15(2):fou005.

[0446] In other embodiments, the pH is approximately 5.7-5.9, 5.8-6.0, 5.9-6.1, 6.0-6.2, 6.1-6.3, 6.2-6.5, 6.4-6.7, 6.5-6.8, 6.6-6.9, 6.7-7.0, 6.8-7.1, 6.9-7.2, 7.0-7.3, 7.1-7.4, 7.2-7.5, 7.3-7.6, 7.4-7.7, 7.5-7.8, 7.6-7.9, 7.7-8.0, 7.8-8.1, 7.9-8.2, 8.0-8.3, 8.1-8.4, 8.2-8.5, 8.3-8.6, 8.4-8.7, or 8.5-8.8.

[0447] In some embodiments, the pH of the culture medium may be at least 5.5. In other embodiments, the culture medium may have a pH level of about 5.5. In other embodiments, the culture medium may have a pH level of 4 to 8. In some cases, the culture is maintained at a pH level of 5.5 to 8. In other embodiments, the culture medium has a pH level of 6 to 8. In some cases, the culture medium has a pH level that is maintained at a pH level of 6 to 8. In some embodiments, the yeast is grown and / or maintained at a pH level of 6.1 to 8.1. In some embodiments, the yeast is grown and / or maintained at a pH level of 6.2 to 8.2. In some embodiments, the yeast is grown and / or maintained at a pH level of 6.3 to 8.3. In some embodiments, the yeast is grown and / or maintained at a pH level of 6.4 to 8.4. In some embodiments, the yeast is grown and / or maintained at a pH level of 5.5 to 8.5. In some embodiments, the yeast is grown and / or maintained at a pH level of 6.5 to 8.5. In some embodiments, the yeast is grown at a pH level of approximately 5.6, 5.7, 5.8, or 5.9. In some embodiments, the yeast is grown at a pH level of approximately 6. In some embodiments, the yeast is grown at a pH level of approximately 6.5. In some embodiments, the yeast is grown at a pH level of approximately 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the yeast is grown at a pH level of approximately 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the yeast is grown at a level greater than 8.

[0448] In some embodiments, the pH of the culture medium may be in the range of pH 2 to 8.5. In certain embodiments, the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0449] Exemplary methods for yeast culture can be found in U.S. Patent No. 5,436,136, entitled “Inhibitory Yeast Promoter” (Filing: 12 / 20 / 1991, Assignee: Ciba-Geigy Corporation), U.S. Patent No. 6,645,739, entitled “Yeast Expression System, Method for Producing Polypeptides in Yeast, and Related Compositions” (Filing: 07 / 26 / 2001, Assignee: Phoenix Pharmacologies, Inc., Lexington, KY), and U.S. Patent No. 10,023,836, entitled “Yeast Culture Medium” (Filing: 08 / 23 / 2013, Assignee: Yamaguchi University) (their disclosures are incorporated herein by reference in their entirety).

[0450] fermentation The present invention aims to cultivate host organisms in any form of fermentation. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes can be used herein.

[0451] Fermentation can be carried out on any scale. The methods and techniques intended according to the present invention are useful for recombinant protein expression on any scale. Therefore, in some embodiments, for example, microliter, milliliter, centiliter, and deciliter scale fermentation volumes may be used, as may 1-liter scale and larger fermentation volumes.

[0452] In some embodiments, the fermentation volume is approximately 1 liter or more. For example, in some embodiments, the fermentation volume is approximately 1 liter to approximately 100 liters. In some embodiments, the fermentation volume is approximately 1 liter, approximately 2 liters, approximately 3 liters, approximately 4 liters, approximately 5 liters, approximately 6 liters, approximately 7 liters, approximately 8 liters, approximately 9 liters, or approximately 10 liters. In some embodiments, the fermentation volume is approximately 1 liter to approximately 5 liters, approximately 1 liter to approximately 10 liters, approximately 1 liter to approximately 25 liters, approximately 1 liter to approximately 50 liters, approximately 1 liter to approximately 75 liters, approximately 10 liters to approximately 25 liters, approximately 25 liters to approximately 50 liters, or approximately 50 liters to approximately 100 liters. In other embodiments, the fermentation volume is 5 liters, 10 liters, 15 liters, 20 liters, 25 liters, 50 liters, 75 liters, 100 liters, 200 liters, 500 liters, 1,000 liters, 2,000 liters, 5,000 liters, 10,000 liters, or 50,000 liters or more.

[0453] In some embodiments, the fermentation medium may be a nutrient solution used for cell growth and / or maintenance. This solution typically provides at least one component from one or more of the following categories: (1) an energy source (usually in the form of a carbon source, e.g., glucose), (2) all essential amino acids (usually the basic set of 20 amino acids), (3) vitamins and / or other organic compounds required in low concentrations, (4) free fatty acids or lipids (e.g., linoleic acid), and (5) trace elements (trace elements are typically defined as inorganic compounds or naturally occurring elements required in very low concentrations (usually in the micromolar concentration range)).

[0454] In some embodiments, the fermentation medium may be the same as the cell culture medium described herein or any other medium. In some embodiments, the fermentation medium may be different from the cell culture medium. In some embodiments, the fermentation medium may be modified to suit large-scale protein production.

[0455] In some embodiments, the fermentation medium may be selectively supplemented with one or more components from any of the following categories: (1) hormones and other growth factors (e.g., serum, insulin, transferrin, etc.), (2) salts (e.g., magnesium, calcium, and phosphate), (3) buffers (e.g., HEPES), (4) nucleosides and bases (e.g., adenosine, thymidine, etc.), (5) proteins and tissue hydrolysates (e.g., peptone or peptone mixtures which can be obtained from purified gelatin, plant materials, or animal by-products), (6) antibiotics (e.g., gentamicin), and (7) cytoprotective agents (e.g., Pluronic® polyols).

[0456] In some embodiments, the pH of the fermentation medium can be maintained using pH buffers and methods known to those skilled in the art. pH control during fermentation can also be achieved using aqueous ammonia. In some embodiments, the pH of the fermentation medium will be selected based on the preferred pH of the organism used. Therefore, in some embodiments, depending on the host cell and temperature, the pH may be in the range of about 1 to about 10.

[0457] In some embodiments, the pH of the fermentation medium may be in the range of 2 to 8.5. In certain embodiments, the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0458] In other embodiments, the pH is approximately 5.7-5.9, 5.8-6.0, 5.9-6.1, 6.0-6.2, 6.1-6.3, 6.2-6.5, 6.4-6.7, 6.5-6.8, 6.6-6.9, 6.7-7.0, 6.8-7.1, 6.9-7.2, 7.0-7.3, 7.1-7.4, 7.2-7.5, 7.3-7.6, 7.4-7.7, 7.5-7.8, 7.6-7.9, 7.7-8.0, 7.8-8.1, 7.9-8.2, 8.0-8.3, 8.1-8.4, 8.2-8.5, 8.3-8.6, 8.4-8.7, or 8.5-8.8.

[0459] In some embodiments, for example, when Escherichia coli (E. coli) is used, the optimal pH range is 6.5 to 7.5, depending on the temperature.

[0460] In other embodiments, for example, when a yeast strain is used, the pH may be in the range of about 4.0 to 8.0.

[0461] In some embodiments, a neutral pH (i.e., a pH of about 7.0) can be used.

[0462] Those skilled in the art will recognize that during fermentation, the pH level may drift as a result of the conversion and generation of substrates and metabolites.

[0463] In some embodiments, the fermentation medium may be supplemented with buffers or other chemicals to avoid changes in pH. For example, in some embodiments, Ca(OH)2, CaCO3, NaOH, or NH4OH may be added to the fermentation medium to neutralize the formation of acidic compounds that occur in some yeast species during industrial processes.

[0464] Temperature is another important consideration in the fermentation process, and, like pH considerations, temperature depends on the type of host cell selected.

[0465] In some embodiments, the fermentation temperature is maintained between approximately 4°C and approximately 42°C. In certain embodiments, the fermentation temperature is approximately 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C.

[0466] In other embodiments, the fermentation temperature is approximately 25°C to 27°C, 25°C to 28°C, 25°C to 29°C, 25°C to 30°C, 25°C to 31°C, 25°C to 32°C, 25°C to 33°C, 26°C to 28°C, 26°C to 29°C, 26°C to 30°C, 26°C to 31°C, 26°C to 32°C, 27°C to 29°C, 27°C to 30°C, The temperature is maintained at approximately 27°C to 31°C, approximately 27°C to 32°C, approximately 26°C to 33°C, approximately 28°C to 30°C, approximately 28°C to 31°C, approximately 28°C to 32°C, approximately 29°C to 31°C, approximately 29°C to 32°C, approximately 29°C to 33°C, approximately 30°C to 32°C, approximately 30°C to 33°C, approximately 31°C to 33°C, approximately 31°C to 32°C, approximately 30°C to 33°C, or approximately 32°C to 33°C.

[0467] In other embodiments, the temperature changes during fermentation, for example, depending on the stage of fermentation.

[0468] Fermentation can be achieved with a variety of microorganisms known to those skilled in the art. Suitable microorganisms for the scaled-up production of CRIP, CRIP-insecticidal protein, or peptide-IA include any of the microorganisms listed herein. In some embodiments, non-limiting examples of microorganisms include species of the genera Saccharomyces (including, but not limited to, S. cerevisiae (baker's yeast), S. distaticus, and S. uvarum), Clibellomyces (including, but not limited to, K. marxianus and K. fragilis), Candida (including, but not limited to, C. pseudotropicalis and C. brassicae), Pichia stipitis (a close relative of Candida shehatae), Clavispora (including, but not limited to, C. lusitaniae and C. opuntiae), Paxolene (including, but not limited to, P. tannophilus), and Brettanomyces (including, but not limited to, B. clausenii).Other suitable microorganisms include, for example, Zymomonas mobilis, Clostridium spp. (including, but not limited to, C. thermocellum, C. saccharobutylacetonicum, C. saccharobutylicum, C. Puniceum, C. beijernckii, and C. acetobutylicum), Moniliella pollinis, Moniliella megachiliensis, Lactobacillus spp., Yarrowia lipolytica, Aureobasidium sp., Trichosporonoides sp., Trigonopsis variabilis, Trichosporon sp., Moniliella acetoabutans sp., Typhula variabilis, Candida magnolias, Ustilaginomycetes sp., and Pseudozyma. Examples include *Tsukubaensis*, yeast species (Zygosaccharomyces, Debaryomyces, Hansenula, and Pichia genera), and fungi of the *Dematioid* genus, *Torula*. See, for example, Philippidis, GP, 1996, *Cellulose bioconversion technology*, in *Handbook on Bioethanol: Production and Utilization*, Wyman, CE, ed., Taylor & Francis, Washington, DC, 179-212.

[0469] The fermentation medium may be selected according to the needs of the host cells and / or the end user. Any necessary auxiliary agents other than carbon may also be included in appropriate concentrations and introduced alone or in mixtures with other auxiliary agents or media, such as a complex nitrogen source.

[0470] Yeast fermentation Methods of fermentation using yeast are well known to those skilled in the art. In some embodiments, batch fermentation can be used according to the method provided herein, and in other embodiments, a continuous fermentation procedure can be used.

[0471] In some embodiments, a batch fermentation method can be used to produce CRIP, CRIP-insecticidal protein, or peptide-IA of the present invention. Briefly, a batch fermentation method refers to a type of fermentation carried out using a closed system, where the composition of the culture medium is determined at the start of fermentation and is not subjected to artificial changes during fermentation (i.e., the medium is inoculated with one or more yeast cells at the start of fermentation, and fermentation proceeds without interruption by the user). Typically, in a batch fermentation system, the system's metabolites and biomass composition are constantly changing until fermentation stops. In batch culture, yeast cells go through a static induction phase, then a logarithmic phase of high growth, and finally a quiescent phase where the growth rate decreases or stops. If left unchecked, quiescent yeast cells will eventually die. In the batch method, logarithmic phase yeast cells generally carry out the majority of the synthesis of the final product.

[0472] In some embodiments, fed-batch fermentation can be used to produce CRIP, CRIP-insecticidal protein, or peptide-IA of the present invention. Briefly, fed-batch fermentation is similar to a typical batch method (described above), except that in the fed-batch method, the substrate is gradually added as fermentation progresses. Fed-batch fermentation is useful when catabolism inhibition can inhibit the metabolism of yeast cells and when it is desirable to have a limited amount of substrate in the culture medium. Generally, the measurement of substrate concentration in a fed-batch system is estimated based on changes in measurable factors that reflect metabolism, such as pH, dissolved oxygen, and partial pressure of exhaust gases (e.g., CO2).

[0473] In some embodiments, a fed-batch fermentation procedure can be used to produce CRIP, CRIP-insecticidal protein, or peptide-IA as follows: Culturing the product organism (e.g., modified yeast cells) in a 10 L bioreactor spurged with an N2 / CO2 mixture using 5 L of broth containing 5 g / L potassium phosphate, 2.5 g / L ammonium chloride, 0.5 g / L magnesium sulfate, and 30 g / L corn steep liquor, as well as a first carbon source and a second carbon source at concentrations of 20 g / L. As the modified yeast cells grow and utilize the carbon source, an additional 70% carbon source mixture is then supplied to the bioreactor at a rate roughly balanced with the consumption of the carbon source. The bioreactor temperature is generally maintained at 30°C. Growth continues for about 24 hours or longer until the heterologous peptide reaches the desired concentration (e.g., cell density is about 5-10 g / L). Once the culture period is complete, the contents of the fermenter can be passed through a cell separation unit such as a centrifuge to remove cells and cell debris, and the fermentation broth can be transferred to a product separation unit. Isolation of heterologous peptides can be performed by standard separation procedures well known in the art.

[0474] In some embodiments, continuous fermentation can be used to produce CRIP, CRIP-insecticidal protein, or peptide-IA of the present invention. Briefly, continuous fermentation refers to fermentation using an open system, in which fermentation medium is continuously added to a bioreactor and processed by simultaneously removing approximately equal amounts of conditional medium. Continuous fermentation generally maintains the culture at high density, and the yeast cells are mainly in the logarithmic growth phase. Typically, the continuous fermentation method is performed to maintain steady-state growth conditions, and the loss of yeast cells due to medium recovery must be balanced with the cell growth rate in fermentation.

[0475] In some embodiments, CRIP, CRIP-insecticide protein, or peptide-IA can be produced using a continuous fermentation method as follows: Modified yeast strains can be cultured using a bioreactor apparatus and culture medium composition, where the first and second carbon sources are, for example, about 30-50 g / L. When the carbon sources are depleted, feed medium of the same composition is continuously supplied at a rate of about 0.5 L / hour to 1 L / hour, and the liquid is recovered at the same rate. The concentration of heterologous peptides in the bioreactor remains generally constant along with the cell density. The temperature is generally maintained at 30°C, and the pH is generally maintained at about 4.5 using concentrated NaOH and concentrated HCl as needed.

[0476] In some embodiments, when producing CRIP, CRIP-insecticidal protein, or peptide-IA, the bioreactor can be operated continuously for, for example, about one month, with samples taken daily or as needed to ensure consistency in the concentration of the target compound. In continuous mode, the contents of the fermenter are constantly removed as fresh culture medium is supplied. The outlet stream, containing cells, medium, and heterologous peptides, can then be ...

Claims

1. A combination containing cysteine-rich insecticidal peptide (CRIP) and insecticide (IA).

2. The combination according to claim 1, wherein IA is a bacterial toxin, a fungal toxin, a lectin, an Azadirachta indica compound, a boron compound, a virus, or a combination thereof.

3. The combination according to claim 2, wherein the bacterial toxin is Bacillus thuringiensis (Bt) toxin or Photorhabdus toxin.

4. The combination according to claim 3, wherein the Bt toxin is one or more fermentation solids, spores, or toxins isolated from the group consisting of: Bacillus thuringiensis var. kurstaki (Btk), Bacillus thuringiensis var. tenebrionis (Btt), Bacillus thuringiensis var. israelensis (Bti), Bacillus thuringiensis var. aizawai, Bacillus thuringiensis var. aizawai / pacificus, Bacillus thuringiensis var. alesti, Bacillus thuringiensis var. amagiensis, Bacillus thuringiensis var. andalousiensis, Bacillus thuringiensis var. argentensis, Bacillus thuringiensis var. asturiensis, Bacillus thuringiensis var. azorensis, Bacillus thuringiensis var. balearica, Bacillus thuringiensis var. berliner, Bacillus thuringiensis var. bolivia, Bacillus thuringiensis var. brasiliensis, Bacillus thuringiensis var. cameroun, Bacillus thuringiensis var. canadensis, Bacillus thuringiensis var. chanpaisis, Bacillus thuringiensis var. chinensis, Bacillus thuringiensis var. colmeri, Bacillus thuringiensis var. koreensis, Bacillus thuringiensis var. dakota, Bacillus thuringiensis var. darmstadienis, Bacillus thuringiensis var. dendrolimus, Bacillusthuringiensis var. entomocidus, Bacillus thuringiensis var. entomocidus / subtoxicus, Bacillus thuringiensis var. finitimus, Bacillus thuringiensis var. fukuokaensis, Bacillus thuringiensis var. galechiae, Bacillus thuringiensis var. galleryae, Bacillus thuringiensis var. graciosiensis, Bacillus thuringiensis var. guiyangensis, Bacillus thuringiensis var. higo, Bacillus thuringiensis var. huazhongensis, Bacillus thuringiensis var. iberica, Bacillus thuringiensis var. indiana, Bacillus thuringiensis var.israelensis / tochigiensis、｢bacillus thuringiensis var.japonensis、｢bacillus thuringiensisvvar.jegathesan、｢bacillus thuringiensis vireesssstreressss vinnenmmemmemerononsnn vi.EEE27、Bacillus thuringiensis vir.erereresvar. londrina, Bacillus thuringiensis var. malayensis, Bacillus thuringiensis var. medellin, Bacillus thuringiensis var. mexicanensis, Bacillus thuringiensis var. mogi, Bacillus thuringiensis var. monterrey, Bacillus thuringiensis var. morrisoni, Bacillus thuringiensis var.muju、Bacillus thurssimiivar.navarreis、Bacillustheureiis var.nirimiis、Bacillus thursisiis var.novosiirrk、Bacillustiurriin var.oswaldocruzi、Bacillus thuringiensis var. pahangi, Bacillus thuringiensis var. pakistani, Bacillus thuringiensis var. palmanyolensis, Bacillus thuringiensis var. pingluonsis, Bacillus thuringiensis var. pirenaica, Bacillus thuringiensis var. poloniensis, Bacillus thuringiensis var.pimichimiis、Bacillus thurssimis var.piimmis、Bacilmistiar. var.roskildimis、Bacillus thursssiis var.seieeo、Bacillustheuresisvar. shandongiensis, Bacillus thuringiensis var. silo, Bacillus thuringiensis var. sinensis, Bacillus thuringiensis var. sooncheon, Bacillus thuringiensis var. sotto, Bacillus thuringiensis var. sotto / dendrolimus, Bacillus thuringiensis var. subtoxicus, Bacillus thurimimiis var.sumiyoshiiis、Bacillus thurimir. thurimiiis var.thompsii、Bacillus thurimar.thurimiitin thurorimiis var.toruchii、Bacillus thurotiritooioouiistiirn var.toummanooffi、Bacillus thurssisiivar.viueis、Bacillis var. wuhanensis, Bacillus thuringiensis var. xiaguangiensis, Bacillus thuringiensis var. yosoo, Bacillus thuringiensis var. yunnanensis, Bacillus thuringiensis var. zhaodongensis, and Bacillus thuringiensis var. konkukian toxins.

5. The combination according to claim 4, wherein the Bt toxin is one or more fermented solids, spores, or toxins isolated from the group consisting of: Bacillus thuringiensis var. kurstaki (Btk), Bacillus thuringiensis var. tenebrionis (Btt), and Bacillus thuringiensis var. israelensis (Bti).

6. The combination according to claim 5, wherein the Bt toxin is a parasporal crystalline toxin, a secreted protein, a β-exotoxin, a 41.9-kDa insecticide, a sphericolylinsin, an alveolylinsin, or an enhancenin-like protein.

7. The combination according to claim 6, wherein the paraspore crystalline toxin is δ-endotoxin.

8. The combination according to claim 7, wherein the δ-endotoxin is a 3-domain (3D) Cry family protein, a binary Bin-like family toxin, an ETX_MTX2-like family toxin, a Toxin-10 family toxin, an erolysine family toxin, or a cytolysin.

9. The combination according to claim 8, wherein the δ-endotoxin is a 3-domain (3D) Cry toxin, a mosquito-killing Cry toxin (Mtx), a binary-like (Bin) toxin, or a Cyt toxin.

10. The combination according to claim 9, wherein the δ-endotoxin is a 3-domain (3D) Cry toxin or Cyt toxin.

11. The combination according to claim 10, wherein the δ-endotoxin is selected from the group consisting of: Cry1Aa1, Cry1Aa2, Cry1Aa3, Cry1Aa4, Cry1Aa5, Cry1Aa6, Cry1Aa7, Cry1Aa8, Cry1Aa9, Cry1Aa10, Cry1Aa11, Cry1Aa12, Cry1Aa13, Cry1Aa14, Cry1Aa15, Cry1Aa16, Cry1Aa17, Cry1Aa18, Cry1Aa19, Cry1Aa20, Cry1Aa21, Cry1Aa22, Cry1Aa23, Cry1Aa24 , Cry1Aa25, Cry1Ab1, Cry1Ab2, Cry1Ab3, Cry1Ab4, Cry1Ab5, Cry1Ab6, Cry1 Ab7, Cry1Ab8, Cry1Ab9, Cry1Ab10, Cry1Ab11, Cry1Ab12, Cry1Ab13, Cry1Ab 14, Cry1Ab15, Cry1Ab16, Cry1Ab17, Cry1Ab18, Cry1Ab19, Cry1Ab20, Cry1A b21, Cry1Ab22, Cry1Ab23, Cry1Ab24, Cry1Ab25, Cry1Ab26, Cry1Ab27, Cry1A b28, Cry1Ab29, Cry1Ab30, Cry1Ab31, Cry1Ab32, Cry1Ab33, Cry1Ab34, Cry1Ab35, Cry1Ab36, Cry1Ab-sama, Cry1Ab-sama, Cry1Ab-sama, Cry1Ab-sama, Cry1Ac1, Cry1Ac2, Cry1Ac3, Cry1Ac4, Cry1Ac5, Cry1Ac6, Cry1Ac7, Cry1Ac8, Cry1Ac9, Cry1Ac 10, Cry1Ac11, Cry1Ac12, Cry1Ac13, Cry1Ac14, Cry1Ac15, Cry1Ac16, Cry1Ac 17, Cry1Ac18, Cry1Ac19, Cry1Ac20, Cry1Ac21, Cry1Ac22, Cry1Ac23, Cry1A c24, Cry1Ac25, Cry1Ac26, Cry1Ac27, Cry1Ac28, Cry1Ac29, Cry1Ac30, Cry1 Ac31, Cry1Ac32, Cry1Ac33, Cry1Ac34, Cry1Ac35, Cry1Ac36, Cry1Ac37, Cry 1Ac38, Cry1Ac39, Cry1Ad1, Cry1Ad2, Cry1Ae1, Cry1Af1, Cry1Ag1, Cry1Ah1,Cry1Ah2, Cry1Ah3, Cry1Ai1, Cry1Ai2, Cry1Aj1, Cry1A-sama, Cry1Ba1, Cry1Ba2, Cry1Ba3, Cry1Ba4, Cry1Ba5, Cry1Ba6, Cry1Ba7, Cry1Ba8, Cry1Bb1, Cry1B b2, Cry1Bb3, Cry1Bc1, Cry1Bd1, Cry1Bd2, Cry1Bd3, Cry1Be1, Cry1Be2, Cry 1Be3, Cry1Be4, Cry1Be5, Cry1Bf1, Cry1Bf2, Cry1Bg1, Cry1Bh1, Cry1Bi1, C ry1Bj1, Cry1Ca1, Cry1Ca2, Cry1Ca3, Cry1Ca4, Cry1Ca5, Cry1Ca6, Cry1Ca7 , Cry1Ca8, Cry1Ca9, Cry1Ca10, Cry1Ca11, Cry1Ca12, Cry1Ca13, Cry1Ca14, Cry1Ca15, Cry1Cb1, Cry1Cb2, Cry1Cb3, Cry1Cb-like, Cry1Da1, Cry1Da2, Cry1D a3, Cry1Da4, Cry1Da5, Cry1Db1, Cry1Db2, Cry1Dc1, Cry1Dd1, Cry1Ea1, Cry 1Ea2, Cry1Ea3, Cry1Ea4, Cry1Ea5, Cry1Ea6, Cry1Ea7, Cry1Ea8, Cry1Ea9, C ry1Ea10, Cry1Ea11, Cry1Ea12, Cry1Eb1, Cry1Fa1, Cry1Fa2, Cry1Fa3, Cry1 Fa4, Cry1Fb1, Cry1Fb2, Cry1Fb3, Cry1Fb4, Cry1Fb5, Cry1Fb6, Cry1Fb7, Cr y1Ga1, Cry1Ga2, Cry1Gb1, Cry1Gb2, Cry1Gc1, Cry1Ha1, Cry1Hb1, Cry1Hb2, Cry1Hc1, Cry1H-like, Cry1Ia1, Cry1Ia2, Cry1Ia3, Cry1Ia4, Cry1Ia5, Cry1Ia6 , Cry1Ia7, Cry1Ia8, Cry1Ia9, Cry1Ia10, Cry1Ia11, Cry1Ia12, Cry1Ia13, C ry1Ia14, Cry1Ia15, Cry1Ia16, Cry1Ia17, Cry1Ia18, Cry1Ia19, Cry1Ia20, Cry1Ia21, Cry1Ia22, Cry1Ia23, Cry1Ia24, Cry1Ia25, Cry1Ia26, Cry1Ia27,Cry1Ia28, Cry1Ia29, Cry1Ia30, Cry1Ia31, Cry1Ia32, Cry1Ia33, Cry1Ia34 , Cry1Ia35, Cry1Ia36, Cry1Ia37, Cry1Ia38, Cry1Ia39, Cry1Ia40, Cry1Ib1 , Cry1Ib2, Cry1Ib3, Cry1Ib4, Cry1Ib5, Cry1Ib6, Cry1Ib7, Cry1Ib8, Cry1I b9, Cry1Ib10, Cry1Ib11, Cry1Ic1, Cry1Ic2, Cry1Id1, Cry1Id2, Cry1Id3, Cr y1Ie1, Cry1Ie2, Cry1Ie3, Cry1Ie4, Cry1Ie5, Cry1If1, Cry1Ig1, Cry1I-sama, Cry1I-sama, Cry1Ja1, Cry1Ja2, Cry1Ja3, Cry1Jb1, Cry1Jc1, Cry1Jc2, Cry1Jd1, C ry1Ka1, Cry1Ka2, Cry1La1, Cry1La2, Cry1La3, Cry1Ma1, Cry1Ma2, Cry1Na1, Cry1Na2, Cry1Na3, Cry1Nb1, Cry1-sama, Cry2Aa1, Cry2Aa2, Cry2Aa3, Cry2Aa4, Cry2Aa5, Cry2Aa6, Cry2Aa7, Cry2Aa8, Cry2Aa9, Cry2Aa10, Cry2Aa11, Cry2 Aa12, Cry2Aa13, Cry2Aa14, Cry2Aa15, Cry2Aa16, Cry2Aa17, Cry2Aa18, Cry 2Aa19, Cry2Aa20, Cry2Aa21, Cry2Aa22, Cry2Aa23, Cry2Aa23, Cry2Aa25, Cr y2Ab1, Cry2Ab2, Cry2Ab3, Cry2Ab4, Cry2Ab5, Cry2Ab6, Cry2Ab7, Cry2Ab8, C ry2Ab9, Cry2Ab10, Cry2Ab11, Cry2Ab12, Cry2Ab13, Cry2Ab14, Cry2Ab15, C ry2Ab16, Cry2Ab17, Cry2Ab18, Cry2Ab19, Cry2Ab20, Cry2Ab21, Cry2Ab22, C ry2Ab23, Cry2Ab24, Cry2Ab25, Cry2Ab26, Cry2Ab27, Cry2Ab28, Cry2Ab29, Cry2Ab30, Cry2Ab31, Cry2Ab32, Cry2Ab33, Cry2Ab34, Cry2Ab35, Cry2Ab36,Cry2Ac1, Cry2Ac2, Cry2Ac3, Cry2Ac4, Cry2Ac5, Cry2Ac6, Cry2Ac7, Cry2Ac 8, Cry2Ac9, Cry2Ac10, Cry2Ac11, Cry2Ac12, Cry2Ad1, Cry2Ad2, Cry2Ad3, C ry2Ad4, Cry2Ad5, Cry2Ae1, Cry2Af1, Cry2Af2, Cry2Ag1, Cry2Ah1, Cry2Ah2 , Cry2Ah3, Cry2Ah4, Cry2Ah5, Cry2Ah6, Cry2Ai1, Cry2Aj1, Cry2Ak1, Cry2Al 1, Cry2Ba1, Cry2Ba2, Cry3Aa1, Cry3Aa2, Cry3Aa3, Cry3Aa4, Cry3Aa5, Cry3 Aa6, Cry3Aa7, Cry3Aa8, Cry3Aa9, Cry3Aa10, Cry3Aa11, Cry3Aa12, Cry3Ba1 , Cry3Ba2, Cry3Ba3, Cry3Bb1, Cry3Bb2, Cry3Bb3, Cry3Ca1, Cry4Aa1, Cry4Aa2, Cry4Aa3, Cry4Aa4, Cry4A-sama, Cry4Ba1, Cry4Ba2, Cry4Ba3, Cry4Ba4, Cry4B a5, Cry4Ba-like, Cry4Ca1, Cry4Ca2, Cry4Cb1, Cry4Cb2, Cry4Cb3, Cry4Cc1, Cry 5Aa1, Cry5Ab1, Cry5Ac1, Cry5Ad1, Cry5Ba1, Cry5Ba2, Cry5Ba3, Cry5Ca1, C ry5Ca2, Cry5Da1, Cry5Da2, Cry5Ea1, Cry5Ea2, Cry6Aa1, Cry6Aa2, Cry6Aa3 , Cry6Ba1, Cry7Aa1, Cry7Aa2, Cry7Ab1, Cry7Ab2, Cry7Ab3, Cry7Ab4, Cry7Ab 5, Cry7Ab6, Cry7Ab7, Cry7Ab8, Cry7Ab9, Cry7Ac1, Cry7Ba1, Cry7Bb1, Cry7 Ca1, Cry7Cb1, Cry7Da1, Cry7Da2, Cry7Da3, Cry7Ea1, Cry7Ea2, Cry7Ea3, Cry 7Fa1, Cry7Fa2, Cry7Fb1, Cry7Fb2, Cry7Fb3, Cry7Ga1, Cry7Ga2, Cry7Gb1, C ry7Gc1, Cry7Gd1, Cry7Ha1, Cry7Ia1, Cry7Ja1, Cry7Ka1, Cry7Kb1, Cry7La1,Cry8Aa1, Cry8Ab1, Cry8Ac1, Cry8Ad1, Cry8Ba1, Cry8Bb1, Cry8Bc1, Cry8Ca 1, Cry8Ca2, Cry8Ca3, Cry8Ca4, Cry8Ca5, Cry8Da1, Cry8Da2, Cry8Da3, Cry8 Db1, Cry8Ea1, Cry8Ea2, Cry8Ea3, Cry8Ea4, Cry8Ea5, Cry8Ea6, Cry8Fa1, Cr y8Fa2, Cry8Fa3, Cry8Fa4, Cry8Ga1, Cry8Ga2, Cry8Ga3, Cry8Ha1, Cry8Hb1, C ry8Ia1, Cry8Ia2, Cry8Ia3, Cry8Ia4, Cry8Ib1, Cry8Ib2, Cry8Ib3, Cry8Ja1 , Cry8Ka1, Cry8Ka2, Cry8Ka3, Cry8Kb1, Cry8Kb2, Cry8Kb3, Cry8La1, Cry8Ma 1. Cry8Ma2, Cry8Ma3, Cry8Na1, Cry8Pa1, Cry8Pa2, Cry8Pa3, Cry8Qa1, Cry8Qa2, Cry8Ra1, Cry8Sa1, Cry8Ta1, Cry8-sama, Cry8-sama, Cry9Aa1, Cry9Aa2, Cry9Aa3 , Cry9Aa4, Cry9Aa5, Cry9Aa, like, Cry9Ba1, Cry9Ba2, Cry9Bb1, Cry9Ca1, C ry9Ca2, Cry9Cb1, Cry9Da1, Cry9Da2, Cry9Da3, Cry9Da4, Cry9Db1, Cry9Dc1 , Cry9Ea1, Cry9Ea2, Cry9Ea3, Cry9Ea4, Cry9Ea5, Cry9Ea6, Cry9Ea7, Cry9E a8, Cry9Ea9, Cry9Ea10, Cry9Ea11, Cry9Eb1, Cry9Eb2, Cry9Eb3, Cry9Ec1, Cr y9Ed1, Cry9Ee1, Cry9Ee2, Cry9Fa1, Cry9Ga1, Cry9-sama, Cry10Aa1, Cry10Aa2, Cry10Aa3, Cry10Aa4, Cry10A-sama, Cry11Aa1, Cry11Aa2, Cry11Aa3, Cry11Aa4, C ry11Aa5, Cry11Aa, Cry11Ba1, Cry11Bb1, Cry11Bb2, Cry12Aa1, Cry13Aa1, Cry13Aa2, Cry14Aa1, Cry14Ab1, Cry15Aa1, Cry16Aa1, Cry17Aa1, Cry18Aa1,Cry18Ba1, Cry18Ca1, Cry19Aa1, Cry19Ba1, Cry19Ca1, Cry20Aa1, Cry20Ba1, Cry20Ba2, Cry20, Cry21Aa1, Cry21Aa2, Cry21Aa3, Cry21Ba1, Cry21Ca1, Cry21Ca2, Cry21, Da1、Cry21Ea1、Cry21Fa1、Cry21Ga1、Cry21Ha1、Cry22Aa1、Cry22Aa2、Cry22Aa3、Cry22Ab1、Cry22Ab2、Cry22Ba1、Cry22Bb1、Cry23Aa1、Cry24Aa1、Cry24Ba1、Cry24Ca1、Cry24Da1、Cry25Aa1、Cry26Aa1、Cry27Aa1、Cry28Aa1、Cry28Aa2、Cry29Aa1、Cry29Ba1、Cry30Aa1、Cry30Ba1、Cry30Ca1、Cry30Ca2、Cry30Da1、Cry30Db1、Cry30Ea1、Cry30Ea2、Cry30Ea3、Cry30Ea4、Cry30Fa1、Cry30Ga1、Cry30Ga2、Cry31Aa1、Cry31Aa2、Cry31Aa3、Cry31Aa4、Cry31Aa5、Cry31Aa6、Cry31Ab1、Cry31Ab2、Cry31Ac1、Cry31Ac2、Cry31Ad1、Cry31Ad2、Cry32Aa1、Cry32Aa2、Cry32Ab1、Cry32Ba1、Cry32Ca1、Cry32Cb1、Cry32Da1、Cry32Ea1、Cry32Ea2、Cry32Eb1、Cry32Fa1、Cry32Ga1、Cry32Ha1、Cry32Hb1、Cry32Ia1、Cry32Ja1、Cry32Ka1、Cry32La1、Cry32Ma1、Cry32Mb1、Cry32Na1、Cry32Oa1、Cry32Pa1、Cry32Qa1、Cry32Ra1、Cry32Sa1、Cry32Ta1、Cry32Ua1、Cry32Va1、Cry32Wa1、Cry32Wa2、Cry32Xa1、Cry32Ya1、Cry33Aa1、Cry34Aa1、Cry34Aa2、Cry34Aa3、Cry34Aa4、Cry34Ab1、Cry34Ac1、Cry34Ac2、Cry34Ac3、Cry34Ba1、Cry34Ba2、Cry34Ba3、Cry35Aa1、Cry35Aa2、Cry35Aa3、Cry35Aa4、Cry35Ab1、Cry35Ab2、Cry35Ab3、Cry35Ac1、Cry35Ba1、Cry35Ba2、Cry35Ba3、Cry36Aa1、Cry37Aa1、Cry38Aa1、Cry39Aa1、Cry40Aa1、Cry40Ba1、Cry40Ca1, Cry40Da1, Cry41Aa1, Cry41Ab1, Cry41Ba1, Cry41Ba2, Cry41Ca1 , Cry42Aa1, Cry43Aa1, Cry43Aa2, Cry43Ba1, Cry43Ca1, Cry43Cb1, Cry43Cc1 , Cry43, Cry44Aa1, Cry45Aa1, Cry45Ba1, Cry46Aa1, Cry46Aa2, Cry46Ab1, Cry47Aa1, Cry48Aa1, Cry48Aa2, Cry48Aa3, Cry48Ab1, Cry48Ab2, Cry49Aa1, Cry49Aa2, Cry49Aa3, Cry49Aa4, Cry49Ab1, Cry50Aa1, Cry50Ba1, Cry50Ba2 , Cry51Aa1, Cry51Aa2, Cry52Aa1, Cry52Ba1, Cry52Ca1, Cry53Aa1, Cry53Ab1 , Cry54Aa1, Cry54Aa2, Cry54Ab1, Cry54Ba1, Cry54Ba2, Cry55Aa1, Cry55Aa 2, Cry55Aa3, Cry56Aa1, Cry56Aa2, Cry56Aa3, Cry56Aa4, Cry57Aa1, Cry57Ab 1, Cry58Aa1, Cry59Ba1, Cry59Aa1, Cry60Aa1, Cry60Aa2, Cry60Aa3, Cry60B a1, Cry60Ba2, Cry60Ba3, Cry61Aa1, Cry61Aa2, Cry61Aa3, Cry62Aa1, Cry63A a1, Cry64Aa1, Cry64Ba1, Cry64Ca1, Cry65Aa1, Cry65Aa2, Cry66Aa1, Cry66 Aa2, Cry67Aa1, Cry67Aa2, Cry68Aa1, Cry69Aa1, Cry69Aa2, Cry69Ab1, Cry70 Aa1, Cry70Ba1, Cry70Bb1, Cry71Aa1, Cry72Aa1, Cry72Aa2, Cry73Aa1, Cry74Aa, Cry75Aa1, Cry75Aa2, Cry75Aa3, Cry76Aa1, Cry77Aa1, or Cry78Aa1, Cyt 1Aa1, Cyt1Aa2, Cyt1Aa3, Cyt1Aa4, Cyt1Aa5, Cyt1Aa6, Cyt1Aa7, Cyt1Aa8, Cyt1Aa-sama, Cyt1Ab1, Cyt1Ba1, Cyt1Ca1, Cyt1Da1, Cyt1Da2, Cyt2Aa1, Cyt2Aa2,Cyt2Aa3, Cyt2Aa4, Cyt2Ba1, Cyt2Ba2, Cyt2Ba3, Cyt2Ba4, Cyt2Ba5, Cyt2Ba6, Cyt2Ba7, Cyt2Ba8, Cyt2Ba9, Cyt2Ba10, Cyt2Ba11, Cyt2Ba12, Cyt2Ba13, Cyt2Ba14, Cyt2Ba15, Cyt2Ba16, Cyt2Ba, Cyt2Bb1, Cyt2Bc1, Cyt2B, Cyt2Ca1, and Cyt3Aa1.

12. The combination according to claim 11, wherein the Cry toxin or Cyt toxin has the amino acid sequence according to SEQ ID NOs: 412 to 481.

13. The combination according to claim 6, wherein the Bt toxin is a secreted protein.

14. The combination according to claim 13, wherein the secreted protein is a plant insecticidal protein (Vip), a secretory insecticidal protein (Sip), a Bin-like family protein, or an ETX_MTX2 family protein.

15. The combination according to claim 14, wherein the secreted protein is Vip.

16. The combination according to claim 15, wherein the Vip is a Vip1 family protein, a Vip2 family protein, a Vip3 family protein, or a Vip4 family protein.

17. The combination according to claim 16, wherein the Vip is selected from the group consisting of: Vip1Aa1, Vip1Aa2, Vip1Aa3, Vip1Ab1, Vip1Ac1, Vip1Ad1, Vip1Ba1, Vip1Ba2, Vip1Bb1, Vip1Bb2, Vip1Bb3, Vip1Bc1, Vip1Ca1, Vip1Ca2, Vip1Da1, Vip2Aa1, Vip2Aa2, Vip2Aa3, Vip2Ab1, Vip2Ac1, Vip2Ac2, Vip2Ad1, Vip2Ae1, Vip2Ae2, Vip2Ae3, Vip2Af1, Vip2A f2, Vip2Ag1, Vip2Ag2, Vip2Ba1, Vip2Ba2, Vip2Bb1, Vip2Bb2, Vip2Bb3, Vip 2Bb4, Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vi p3Aa8, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3Aa12, Vip3Aa13, Vip3Aa14, Vip 3Aa15, Vip3Aa16, Vip3Aa17, Vip3Aa18, Vip3Aa19.0, Vip3Aa19, Vip3Aa20, V ip3Aa21, Vip3Aa22, Vip3Aa23, Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3Aa41 , Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Aa45, Vip3Aa46, Vip3Aa47, Vip3Aa48 , Vip3Aa49, Vip3Aa50, Vip3Aa51, Vip3Aa52, Vip3Aa53, Vip3Aa54, Vip3Aa5 5, Vip3Aa56, Vip3Aa57, Vip3Aa58, Vip3Aa59, Vip3Aa60, Vip3Aa61, Vip3Aa6 2, Vip3Aa63, Vip3Aa64, Vip3Aa65, Vip3Aa66, Vip3Ab1, Vip3Ab2, Vip3Ac1, V ip3Ad1, Vip3Ad2, Vip3Ad3, Vip3Ad4, Vip3Ad5, Vip3Ad6, Vip3Ae1, Vip3Af1,- 6. ,Vip3Ag15,Vip3Ah1,Vip3Ah2,Vip3Ai1,Vip3Aj1,Vip3Aj2,Vip3Ba1,Vip3Ba2,Vip3Bb1,Vip3Bb2,Vip3Bb3,Vip3Bc,Vip3Ca1,Vip3Ca2,Vip3Ca3,Vip3Ca4,andVip4Aa1.,

18. The combination according to claim 17, wherein the Vip protein has an amino acid sequence according to the amino acid sequences shown in SEQ ID NOs. 482 to 587.

19. The combination according to claim 3, wherein the bacterial toxin is Photorhabdus toxin.

20. The combination according to claim 19, wherein the Photorhabdus toxin is selected from the group consisting of: Photorhabdus akhurstii toxin, Photorhabdus asymbiotica toxin, Photorhabdus asymbiotica subsp. asymbiotica toxin, Photorhabdus asymbiotica subsp. asymbiotica ATCC 43949 toxin, Photorhabdus australis toxin, Photorhabdus australis DSM 17609 toxin, Photorhabdus bodei toxin, Photorhabdus caribbeanensis toxin, Photorhabdus cinerea toxin, Photorhabdus hainanensis toxin, Photorhabdus heterorhabditis toxin, Photorhabdus kayaii toxin, Photorhabdus khanii toxin, Photorhabdus khanii NC19 toxin, Photorhabdus khanii subsp. guanajuatensis toxin, Photorhabdus kleinii toxin, Photorhabdus laumondii toxin, Photorhabdus laumondii subsp. clarkei toxin, Photorhabdus laumondii subsp. laumondii toxin, Photorhabdus laumondii subsp. laumondii TTO1 toxin, Photorhabdus luminescens toxin, Photorhabdus luminescens BA1 toxin, Photorhabdus luminescens NBAII H75HRPL105 toxin, Photorhabdus luminescens NBAII HiPL101 toxin, Photorhabdus luminescens subsp. luminescens toxin, Photorhabdus luminescens subsp. luminescens ATCC 29999 toxin, Photorhabdus luminescens subsp. mexicana toxin, Photorhabdus luminescenssubsp. sonorensis toxin, Photohabdus namnaonensis toxin, Photohabdus noenieputensis toxin, Photohabdus stackbrandti toxin, Photohabdus tasmanensis toxin, Photohabdus temperata toxin, Photohabdus temperata J3 toxin, Photohabdus temperata subsp. Phorame toxin, Phorabdus thermota subsp. thermota toxin, Phorabdus thermota subsp. thermota M1021 toxin, Phorabdus thermota subsp. thermota Meg1 toxin, Phorabdus thracences toxin, unclassified Phorabdus toxin, Phorabdus sp. toxin, Phorabdus sp. 3014 toxin, Phorabdus sp. 3240 toxin, Photohabdus sp. Az29 toxin, Photohabdus sp. BS21 toxin, Photohabdus sp. CbKj163 toxin, Photohabdus sp. CRCIA-PO1 toxin, Photohabdus sp. ENY toxin, Photohabdus sp. FL2122 toxin, Photohabdus sp. FL480 toxin, Photohabdus sp. FsIw96 toxin, Photohabdus sp. GDd233 toxin, Photohabdus sp. H3086 toxin, Photohabdus sp. H3107 toxin, Photohabdus sp. H3240 toxin, Photohabdus sp. HB301 toxin, Photohabdus sp. HB78 toxin, Photohabdus sp. HB89 toxin, Photohabdus sp. HIT toxin, Photohabdus sp. HO1 toxin, Photohabdus sp. HUG-39 toxin, Photohabdus sp. IT toxin, Photohabdus sp. JUN toxin, Photohabdus sp. KCTs129 toxin, Photohabdus sp. KJ13.1TH toxin, Photharhabdus sp. KJ14.3; TH toxin, Photharhabdus sp. KJ24.5; TH toxin, Photharhabdus sp. KJ29.1; TH toxin, Photharhabdus sp. KJ37.1; TH toxin, Photharhabdus sp. KJ7.1; TH toxin, Photharhabdus sp. KJ8.2; TH toxin, Photharhabdus sp. KJ9.1; TH toxin, Photharhabdus sp. KJ9.2 TH toxin, Photohabdus sp. KK1.3 TH toxin, Photohabdus sp. KK1.4 TH toxin, Photohabdus sp. KMD74 toxin, Photohabdus sp. KOH toxin, Photohabdus sp. MID10 toxin, Photohabdus sp. MOL toxin, Photohabdus sp. MSW_058 toxin, Photohabdus sp. MSW_079 toxin, Photohabdus sp. NK2.1 TH toxin, Photohabdus sp. NK2.5 TH toxin, Photohabdus sp. NnMt2h toxin, Photohabdus sp. NP1 toxin, Photohabdus sp. OH10 toxin, Photohabdus sp. OnIr40 toxin, Photohabdus sp. OnKn2 toxin, Photohabdus sp. PB10.1 TH toxin, Photohabdus sp. PB16.3 TH toxin, Photohabdus sp. PB17.1 TH toxin, Photohabdus sp. PB17.3 TH toxin, Photohabdus sp. PB2.5 TH toxin, Photohabdus sp. PB22.4 TH toxin, Photohabdus sp. PB22.5 TH toxin, Photohabdus sp. PB32.1 TH toxin, Photohabdus sp. PB33.1 TH toxin, Photohabdus sp. PB33.4 TH toxin, Photohabdus sp. PB37.4 TH toxin, Photohabdus sp. PB39.2 TH toxin, Photohabdus sp. PB4.5 TH toxin, Photohabdus sp. PB41.4 TH toxin, Photohabduss. PB45.5 TH toxin, Photohabdus s. PB47.1 TH toxin, Photohabdus s. PB47.3 TH toxin, Photohabdus s. PB5.1 TH toxin, Photohabdus s. PB5.4 TH toxin, Photohabdus s. PB50.4 TH toxin, Photohabdus s. PB51.4 TH toxin, Photohabdus s. PB52.2 TH toxin, Photohabdus s. PB54.4 TH toxin, Photohabdus sp. PB58.2 TH toxin, Photohabdus sp. PB58.4 TH toxin, Photohabdus sp. PB58.5 TH toxin, Photohabdus sp. PB59.2 TH toxin, Photohabdus sp. PB6.5 TH toxin, Photohabdus sp. PB67.2 TH toxin, Photohabdus sp. PB67.4 TH toxin, Photohabdus sp. PB68.1 TH toxin, Photohabdus sp. PB7.5 TH toxin, Photohabdus sp. PB76.1 TH toxin, Photohabdus sp. PB76.4 TH toxin, Photohabdus sp. PB76.5 TH toxin, Photohabdus sp. PB78.2 TH toxin, Photohabdus sp. PB80.3 TH toxin, Photohabdus sp. PB80.4 TH toxin, Photohabdus sp. Pjun toxin, Photohabdus sp. RW14-46 toxin, Photohabdus sp. S10-54 toxin, Photohabdus sp. S12-55 toxin, Photohabdus sp. S14-60 toxin, Photohabdus sp. S15-56 toxin, Photohabdus sp. S5P8-50 toxin, Photohabdus sp. S7-51 toxin, Photohabdus sp. S8-52 toxin, Photohabdus sp. S9-53 toxin, Photohabdus sp. SJ2 toxin, Photohabdus sp. SN259 toxin, Photohabdus sp. SP1.5 TH toxin, Photohabdus sp. SP16.4TH toxin, Photohabdus sp. SP21.5 TH toxin, Photohabdus sp. SP3.4 TH toxin, Photohabdus sp. SP4.5 TH toxin, Photohabdus sp. SP7.3 TH toxin, Photohabdus sp. TyKb140 toxin, Photohabdus sp. UK76 toxin, Photohabdus sp. VMG toxin, Photohabdus sp. WA21C toxin, Photohabdus sp. WkSs43 toxin, Photohabdus sp. Wx13 toxin, Photohabdus sp. X4 toxin, Photohabdus sp. YNb90 toxin, and Photohabdus sp. ZM toxin.

21. The combination according to claim 20, wherein the Photorhabdus toxin is Photorhabdus luminescens toxin.

22. The combination according to claim 2, wherein the mycotoxin is Ascomycete mycotoxin.

23. The combination according to claim 22, wherein the Ascomycete mycotoxin is a Cordycipitaceae mycotoxin.

24. The combination according to claim 23, wherein the Cordycipitaceae mycotoxin is Akanthomyces toxin, Ascopolyporus toxin, Beauveria toxin, Beejasamuha toxin, Cordyceps toxin, Coremiopsis toxin, Engyodontium toxin, Gibellula toxin, Hyperdermium toxin, Insecticola toxin, Isaria toxin, Lecanicillium toxin, Microhilum toxin, Phytocordyceps toxin, Pseudogibellula toxin, Rotiferophora toxin, Simplicillium toxin, or Torrubiella toxin.

25. The combination according to claim 24, wherein the Cordycipitaceae fungal toxin is a Beauveria toxin.

26. Foreword: Beauveria toxin, Beauveria alpha toxin, Beauveria amorpha toxin, Beauveria arenaria toxin, Beauveria asiatica toxin, Beauveria australis toxin, Beauveria bassiana toxin, Cordyceps bassiana toxin, Beauveria brongniaartti toxin, Beauveria brumptti toxin, Beauveria caledonica toxin, Beauveria Chiromensis toxin, Beauveria coccorum toxin, Beauveria cretacea toxin, Beauveria cylindrospora toxin, Beauveria delacroxii toxin, Beauveria densa toxin, Beauveria dependens toxin, Beauveria doryphorae toxin, Beauveria effusa toxin, Beauveria epigaea toxin, Beauveria felina toxin, Beauveria geodes toxin, Beauveria Globulifera toxin, Beauveria heimii toxin, Beauveria hoplocheli toxin, Beauveria kipukae toxin, Beauveria laxa toxin, Beauveria malawiensis toxin, Beauveria medogensis toxin, Beauveria melolonthae toxin, Beauveria nubicola toxin, Beauveria oryzae toxin, Beauveria paradoxa toxin, Beauveria Paranences toxin, Beauveria parasitica toxin, Beauveria petelloti toxin, Beauveria pseudobassiana toxin, Beauveria rileyi toxin, Beauveria rubra toxin, Beauveria shiotae toxin, Beauveria sobolifera toxin, Beauveria spicata toxin, Beauveria stephanoderis toxin, Beauveria sulfurescen toxin, Beauveria Sungitoxin, BeauveriaThe combination according to claim 25, wherein the toxin is tenella toxin, Beauveria tundrensis toxin, Beauveria velata toxin, Beauveria varroae toxin, Beauveria vermiconia toxin, Beauveria vexans toxin, Beauveria viannai toxin, or Beauveria virella toxin.

27. The combination according to claim 2, wherein the lectin is selected from the group consisting of: Galanthus nivalis agglutinin (GNA), Sambucus nigra lectin (SNA), Mackia amurensis-II (MAL-II), Erythrina cristagalli lectin (ECL), Ricinus communis agglutinin-I (RCA), peanut agglutinin (PNA), wheat germ agglutinin (WGA), Griffonia simplicifolia-II (GSL-II), Con A, Lens culinaris agglutinin (LCA), mannose-binding lectin (MBL), BanLec, galectin, Phaseolus vulgaris hemagglutinin (PHA-L), Phaseolus vulgaris hemagglutinin (PHA-E), and Datura stramonium lectin (DSL).

28. The combination according to claim 27, wherein the lectin is GNA.

29. The Azadirachta indica compounds include azadirachtin, azadirradione, azadirradinolide, deacetylgedunin, deacetylazadirachtinol, desfuranozadirradione, epoxyazadirradione, gedunin, mahmoodin, neemfruitin A, neemfruitin B, nimbolid, nimbin, nimoricinol, ointin acetate, sarannin, saranol, alpha-nimolactone, beta-nimolactone, 2',3'-dihydrosarannin, and 3-deacetyl The combination according to claim 2, wherein the combination is sarannin, 6-deacetylninbin, 7-acetyl-16,17-dehydro-16-hydroxyneotriquilenone, 7-benzoylninbosinol, 7-deacetyl-7-benzoylepoxyazaziradione, 7-deacetyl-7-benzoylgedunin, 7-deacetyl-17-epinimolcinol, 15-hydroxyazaziradione, 17-epi-17-hydroxyazaziradione, 17-epiazaziradione, 20,21,22,23-tetrahydro-23-oxoazazirone, 22,23-dihydronimosinol, or 28-deoxonin bolide.

30. The combination according to claim 2, wherein the boron compound is selected from the group consisting of borax, boric acid, disodium octaborate, sodium borate, sodium metaborate, sodium tetraborate decahydrate, boron oxide, boron carbide, boron nitride, boron tribromide, boron trichloride, and boron trifluoride.

31. The combination according to claim 2, wherein the virus is a Baculoviridae virus.

32. The combination according to claim 31, wherein the Baculoviridae virus is a beta-baculovirus.

33. The aforementioned beta-baculoviruses include Adoxophyes orana granulovirus (granulovirus), Agrotis segetum granulovirus, Artogea rapae granulovirus, Pieris brassicae granulovirus, Choristoneura fumiferana granulovirus, Choristoneura occidentalis granulovirus, Clostera anachoreta granulovirus, Clostera anastomosis granulovirus A, Clostera anastomosis granulovirus Henan, Clostera anastomosis granulovirus B, and Cnaphalocrocis. medinalis granulovirus, Cryptophyllia leucotreta granulovirus, Cydia pomonella granulovirus, Cydia pomonella granulosis virus (Mexican isolate), Diatraea saccharalis granulovirus, Epinotia aporema granulovirus, Erinnyis ello granulovirus, Harrisina brillians granulovirus, Helicoverpa armigera granulovirus, Lacanobia oleracea granulovirus, Mocis latipes granulovirus, Mythimna unipuncta granulovirus A, Pseudalatia unipuncta granulovirus, Mythimna unipuncta granulovirus B, Mythimna unipuncta granulovirus, Phthorimaea operculella granulovirus, Plodia interpunctella granulovirus, Plutella xylostella granulovirus, Spodoptera frugiperda granulovirus, Spodoptera litura granulovirus, Trichoplusia ni granulovirus, Trichoplusia ni granulovirus LBIV-12, Xestiac-nigrum granulovirus, unclassified beta-baculovirus, Achaea janata granulovirus, Adoxophyes honmai granulovirus, Agrotis exclamationis granulovirus, Ameria parlorana granulovirus, Andraca bipunctata granulovirus, Autographa gamma granulovirus, Caloptilia theivora granulovirus, Christoneura murinana granulovirus, Christoneura viridis beta-baculovirus, Clostera anastomosis granulovirus, Cnephasia Longana granulovirus, Estigmene acrea granulovirus, Euxoa ochrogaster granulovirus, Heliothis armigera granulovirus, Hoplodrina ambigua granulovirus, Hyphantria cunea granulovirus, Natada nararia granulovirus, Nephelodes emmedonia granulovirus, Pandemis limitata granulovirus, Peridorma morpontora granulovirus, Pieris rapae granulovirus, Plathypena scabra granulovirus, Pseudaletia The combination according to claim 32, wherein the combination is beta-baculovirus, Scotogramma trifolii granulovirus, Spodoptera androgea granulovirus, Spodoptera littoralis granulovirus, Tecia solanivora granulovirus, or Mocis sp. granulovirus.

34. The combination according to claim 1, wherein the IA is selected from the group consisting of Photorhabdus luminescens toxin, Beauveria bassiana toxin, Galanthus nivaris agglutinin (GNA), azadirachtin compounds, boric acid, and Cydia pomonella granulovirus (CpGV).

35. The combination according to claim 34, wherein the Photorhabdus luminescens toxin comprises the Photorhabdus luminescens toxin complex (Tca).

36. The combination according to claim 35, wherein the Tca comprises TcaA protein (SEQ ID NO: 616), TcaB protein (SEQ ID NO: 617), TcaC protein (SEQ ID NO: 618), and TcaZ protein (SEQ ID NO: 619).

37. The combination according to claim 34, wherein the Beauveria bassiana toxin is Beauvericin toxin.

38. where the viewbericin toxin has the chemical formula C ４５ H ５７ N ３ O ９ a viewbericin toxin having, a viewbericin A toxin having the chemical formula C ４６ H ５９ N ３ O<T ９ or a viewbericin B toxin having the chemical formula C ４７ H ６１ N ３ O ９ The combination according to claim 37, which is a viewbericin B toxin having.

39. The combination according to claim 38, wherein the Beauveria bassiana toxin is beauvericin toxin isolated from the ANT-03 spore of the Beauveria bassiana strain.

40. The combination according to claim 34, wherein the GNA has the amino acid sequence shown in SEQ ID NO:

35.

41. The combination according to claim 34, wherein the CpGV is Cydia pomonella granulovirus isolate V22 virus.

42. The combination according to claim 1, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. israelensis (Bti).

43. The combination according to claim 42, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. israelensis strain BMP144.

44. The combination according to claim 1, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk).

45. The combination according to claim 44, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19.

46. The combination according to claim 1, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. tenebrionis (Btt).

47. The combination according to claim 46, wherein the Btt toxin is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. tenebrionis strain NB-176.

48. The combination according to any one of claims 1 to 47, wherein the CRIP is U1-agatoxin-Ta1b peptide, U1-agatoxin-Ta1b variant polypeptide (TVP), anemone toxin, Av3 variant polypeptide (AVP), Phoneutri toxin, or atracotoxin (ACTX).

49. The combination according to claim 48, wherein the U1-agatoxin-Ta1b peptide has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

1.

50. The combination according to claim 49, wherein the U1-agatoxin-Ta1b peptide has an amino acid sequence according to the amino acid sequence shown in SEQ ID NO:

1.

51. The combination according to claim 48, wherein the TVP has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of sequence numbers 2-15, 49-53, 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, and 653-654.

52. The combination according to claim 51, wherein the TVP has an amino acid sequence represented by any one of the amino acid sequences shown in sequence numbers 2-15, 49-53, 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, and 653-654.

53. The combination according to claim 48, wherein the anemone toxin is Av2 toxin or Av3 toxin.

54. The combination according to claim 53, wherein the Av2 toxin has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

588.

55. The combination according to claim 54, wherein the Av2 toxin has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

588.

56. The combination according to claim 53, wherein the Av3 toxin has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

44.

57. The combination according to claim 56, wherein the Av3 toxin has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

44.

58. The combination according to claim 48, wherein the AVP is AVPa peptide, AVPa-C1 peptide, or AVPb peptide.

59. The combination according to claim 58, wherein the AVPa toxin has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

45.

60. The combination according to claim 59, wherein the AVPa toxin has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

45.

61. The combination according to claim 58, wherein the AVPa-C1 toxin has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

46.

62. The combination according to claim 61, wherein the AVPa-C1 toxin has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

46.

63. The combination according to claim 58, wherein the AVPb toxin has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

47.

64. The combination according to claim 63, wherein the AVPb toxin has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

47.

65. The combination according to claim 48, wherein the CRIP is ctenitoxin (CNTX).

66. The combination according to claim 65, wherein the CNTX is Γ-CNTX-Pn1a.

67. The combination according to claim 66, wherein the Γ-CNTX-Pn1a has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

65.

68. The combination according to claim 67, wherein the Γ-CNTX-Pn1a has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

65.

69. The combination according to claim 48, wherein the CRIP is ACTX.

70. The combination according to claim 69, wherein the ACTX is U-ACTX peptide, omega-ACTX peptide, or kappa-ACTX peptide.

71. The combination according to claim 69, wherein the ACTX is U-ACTX-Hv1a, U+2-ACTX-Hv1a, rU-ACTX-Hv1a, rU-ACTX-Hv1b, κ-ACTX-Hv1a, κ+2-ACTX-Hv1a, ω-ACTX-Hv1a, or ω+2-ACTX-Hv1a.

72. The combination according to claim 71, wherein the ACTX has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of sequence numbers 60-64 and 594.

73. The combination according to claim 72, wherein the ACTX has an amino acid sequence represented by any one of the amino acid sequences shown in sequence numbers 60-64 and 594.

74. The combination according to claim 69, wherein the ACTX has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No.

61.

75. The combination according to claim 74, wherein the ACTX has an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

61.

76. The combination according to any one of claims 1 to 48, wherein the CRIP is selected from the group consisting of U1-agatoxin-Ta1b peptide having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, TVP having an amino acid sequence according to any one of SEQ ID NOs: 2-15, 49-53, 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, and 653-654, Av3 variant polypeptide (AVP) having an amino acid sequence shown in SEQ ID NO: 47, Γ-CNTX-Pn1a having an amino acid sequence shown in SEQ ID NO: 65, or U+2-ACTX-Hv1a having an amino acid sequence shown in SEQ ID NO:

61.

77. A combination according to any one of claims 1 to 76, wherein the ratio of IA to CRIP is approximately 10,000:1, 5,000:1, 1,000:1, 500:1, 250:1, 200:1, 100:1, 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 1:1, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 1:100, 1:200, 1:250, 1:500, 1:1,000, 1:5,000, or 1:10,000.

78. The combination according to claim 77, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. Israelensis (Bti), CRIP is ACTX, and the ratio of one or more fermented solids, spores, and toxins isolated from Bacillus thuringiensis var. Israelensis (Bti) to ACTX is about 1:1 to about 1:5000.

79. The combination according to claim 78, wherein the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. israelensis (Bti) to ACTX is approximately 1:4000.

80. The combination according to claim 77, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk), CRIP is ACTX, and the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to ACTX is about 1:1 to about 1:

10.

81. The combination according to claim 80, wherein the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to ACTX is approximately 1:9.

2.

82. The combination according to claim 77, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk), CRIP is AVP, and the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to AVP is about 1:1 to about 1:1.

5.

83. The combination according to claim 82, wherein the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to AVP is about 1:1.

375.

84. The combination according to claim 77, wherein IA is one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. tenebrionis (Btt), CRIP is ACTX, and the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. tenebrionis (Btt) to ACTX is about 1:1 to about 1:

10.

85. The combination according to claim 84, wherein the ratio of one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis var. tenebrionis (Btt) to ACTX is approximately 1:8.

75.

86. A composition comprising the combination according to any one of claims 1 to 82, further comprising an excipient.

87. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a U1-agatoxin-Ta1b peptide having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

1.

88. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and a U1-agatoxin-Ta1b variant polypeptide (TVP) having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

2.

89. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and an Av3 variant polypeptide (AVP) having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

67.

90. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and the Γ-CNTX-Pn1a toxin having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

65.

91. A combination comprising Beauveria bassiana strain ANT-03 spores and U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

61.

92. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. tenebrionis strain NB-176, and the U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

61.

93. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19, and the U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

61.

94. A combination comprising one or more fermented solids, spores, or toxins isolated from Bacillus thuringiensis ssp. israelensis strain BMP144, and the U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

61.

95. A combination comprising Photorhabdus luminescens toxin and ACTX, wherein the Photorhabdus luminescens toxin is a Photorhabdus luminescens toxin complex (Tca) comprising TcaA (SEQ ID NO: 616), TcaB (SEQ ID NO: 617), TcaC (SEQ ID NO: 618), and TcaZ (SEQ ID NO: 619), and the ACTX peptide is U+2-ACTX-Hv1a toxin (SEQ ID NO: 61).

96. A combination comprising Galanthus nivalis agglutinin (GNA) and ACTX, wherein the GNA has the amino acid sequence shown in SEQ ID NO: 35, and the ACTX peptide is a U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO:

61.

97. A combination comprising azadirachtin and ACTX, wherein the azadirachtin has the chemical formula: C ３５ H ４４ O １６ A combination comprising the above, wherein the ACTX is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence shown in Sequence ID No.

61.

98. A combination comprising a boric acid compound and ACTX, wherein the boric acid compound is H ３ BO ３ A combination having the chemical formula, in which the ACTX peptide is a U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO:

61.

99. A combination comprising Cydia pomonella granulovirus (CpGV) and ACTX, wherein the CpGV is Cydia pomonella granulovirus isolate V22 virus, and the ACTX peptide is the U+2-ACTX-Hv1a toxin having the amino acid sequence shown in SEQ ID NO:

61.

100. A method for using a combination according to any one of claims 1 to 99 to control an insect, comprising providing a combination of at least one CRIP and at least one IA, and applying the combination according to any one of claims 1 to 105 to a part of an insect.

101. The method according to claim 100, wherein the insect is selected from the group consisting of: Achema Sphinx Moth (Hornworm) (Eumorpha achemon), Alfalfa Caterpillar (Colias eurytheme), Almond Moth (Caudra cautella), Amorbia Moth (Amorbia humerosana), Army Worm (Spodoptera spp. (e.g., exigua, frugiperda, littoralis), Pseudaletia) (Unicuncta), Artichoke Plum Moth (Platyptilia carduidactyla), Azalea Caterpillar (Datana major), Bagworm (Thyridopteryx), ephemeraeformis), Banana Moth (Hypercompe scribonia), Banana Skipper (Erionota thrax), Blackheaded Budworm (Acleris) Gloverana, California Oakworm (Phrygania californica), Spring Cankerworm (Paleacrita merriccata), Cherry Fruitworm (Grapholita packardi), China Mark Moth (Nymphula stagnata), Citrus Cutworm (Xylomyges curialis), Codling Moth (Cydia) Pomonella, Cranberry Fruitworm (Acrobasis)Vaccinii), Cross-striped Cabbageworm (Evergestis rimosalis), Cutworm (Noctuid species, Agrotis ipsilon), Douglas Fir Tussock Moss (Orgyia pseudotsugata), Ello Moss (Hornworm) (Erinnyis ello), Elm Spanworm (Ennomos subsignaria), European Grapevine Moss (European Grapevine Moth (Lobesia botrana), European Skipper (Thymelicus lineola (Essex Skipper)), Fall Webworm (Melissopus lateferreanus), Filbert Leafroller (Archips rosanus), Fruit Tree Leafroller (Archips argyrospiria), Grape Berry Moth (Paralobesia viteana), Grape Leafroller (Grape Leafroller (Platynota sultana), Grape Leaf Skeletonizer (Harrisina americana (ground only)), Green Cloverworm (Platypena scabra), Green Striped Mapleworm (Dryocampa rubicunda, Gummosos-Batrachedra, Comosae (Hodges)), Gypsy Moth (Lymantria) Dispar), Hemlock Looper (Lambina fiscellaria), Hornworm (Manduca)(sp.), Imported Cabbageworm (Pieris rapae), Io Moth (Automeris io), Jack Pine Budworm (Choristoneura pinus), Light Brown Apple Moth (Epiphyas postvittana), Melon Worm (Diaphania hyalinata), Mimosa Webworm (Homadaula) Anisocentra, Oblique-banded Leafroller (Christoneura rosaceana), Oleander Moss (Syntomeda epilais), Omnivorous Leafroller (Playnota stultana), Omnivorous Looper (Sabulodes aegrotata), Orange Dog (Papilio cresphonetes), Orange Tortrix (Argyrotaenia) Citrana, Oriental Fruit Moth (Grapholita molesta), Peach Twig Borer (Anarsia lineatella), Pine Butterfly (Neophasia menapia), Podworm (Heliocoverpa zea), Redbanded Leafroller (Argyrotaenia velutinana), Redhumped Caterpillar (Schizura) Concinna, Lindworm Complex (Various Leps.), Saddleback Caterpillar (Sibine stimulea)), Saddle Prominent Caterpillar (Heterocampa guttivitta), Saltmarsh Caterpillar (Estigmene acrea), Sod Webworm (Crambus spp.), Spanworm (Ennomos subsignaria), Fall Cankerworm (Alsophylla pometaria), Spruce Budworm (Choristoneura) fumiferana), Tent Caterpillar (Various Lasiocampidae), Thecla-Thecla Basilides (Geyr), Tobacco Hornworm (Manduca sexta), Tobacco Moss (Ephesia elutella), Tufted Apple Budmoss (Platynota idaeusalis), Twig Borer (Anarsia) Lineatella, Variegated Cutworm (Peridroma saucia), Variegated Leafroller (Platynota flavedana), Velvetbean Caterpillar (Anticarsia gemmatalis), Walnut Caterpillar (Datana integrima), Webworm (Hyphantria cunea), Western Tussock Moth (Orgyia) Vetusta, Southern Cornstalk Borer (Diatraea crambidoides), Corn Ear Worm (CornEarworm, Sweet potato weevil, Pepper weevil, Citrus root weevil, Strawberry root weevil, Pecan weevil, Philbert weevil, Ricewater weevil, Alfalfa weevil, Clover weevil, Tee shot - hole bore, Root weevil, Sugarcane Beetle Beetle, Coffee Berry Borer, Annual Bluegrass Weevil (Listronotus maculicollis), Asiatic Garden Beetle (Maladera castanea), European Chafer (Rhizotroqus majalis), Green June Beetle (Cotinis nitida), Japanese Beetle (Popillia japonica), May or June Beetle June beetle (Phyllophaga sp.), Northern masked chafer (Cyclocephala borealis), Oriental beetle (Anomala orientalis), Southern masked chafer (Cyclocephala lurida), Billbug (Curculionoidea), Aedes aegypti, Busseola fusca, Chilo supppressalis, Culex pipiens, CulexQuinquefasciatus, Diabrotica virgifera, Diatraea saccharalis, Helicoverpa armigera, Helicoverpa zea, Heliothis virescens, Leptinotarsa ​​decemlineata, Ostrinia furnacalis, Ostrinia nubilalis, Pectinophora ghossip iella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Trichoplusia ni, and Xanthogaleruca luteola.

102. A method for controlling a Bacillus thuringiensis toxin-resistant insect using a combination according to any one of claims 1 to 99, comprising providing a combination of at least one CRIP and at least one IA, and then applying the combination to a part of an insect.

103. The method according to claim 102, wherein the Bacillus thuringiensis-resistant toxin insect is selected from the group consisting of: Aedes aegypti, Busseoola fusca, Chilo suppressalis, Culex pipiens, Culex qinquefasciatus, Diabrotica virgifera, Diatraea saccharalis, Helicoverpa armigera, Helicoverpa zea, Heliothys virescens, Leptinotarsa ​​decemlineata, Ostrinia furnacalis, Ostrinia Nubilalis, Pectinophora gossypiella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Trichoplusia ni, and Xanthogaleruca luteola.

104. A method for controlling, eliminating, or suppressing pests, comprising applying an effective amount of a combination of the pesticides described in any one of claims 1 to 99 to the part of the pest or to a plant or animal susceptible to attack by the pest.

105. The method according to claim 104, wherein the pest is selected from the group consisting of: ahimas sphinx moss (horn worm) (Eumorpha achemon), alfalfa caterpillar (Colias eurytheme), almond moss (Caudra cautella), amorbia moss (Amorbia humerosana), army worm (Spodoptera spp. (e.g., exigua, frugiperda, littoralis), Pseudaletia unipuncta), artichoke plume moss (Platyptilia carduidactyla), azalea caterpillar (Datana) Major, Bagworm (Thyridopteryx), Ephemeraeformis, Banana Moss (Hypercompe scribonia), Banana Skipper (Erionota thrax), Blackheaded Budworm (Acleris gloverana), California Oakworm (Phrygania californica), Spring Canker Worm (Paleacrita merriccata), Cherry Fruit Worm (Grapholita packardi), China Mark Moss (Nymphula stagnata), Citrus Cut Worm (Xylomyges curialis), Codling Moss (Cydia) Pomonella, Cranberry Fruit Worm (Acrobasis vaccinii), Cross-Striped Cabbage Worm (Evergestis rimosalis), Cut Worm (Noctuid species, Agrotis ipsilon), Douglas Farthenock Moss (Orgyia pseudotsugata), Ello Moss (Horn Worm) (Erinnyis ello), Elm Span Worm (Ennomos subsignaria), European Grapevine Moss (Lobesia botrana), European Skipper (Thymelicus lineola (Essex Skipper)), Fall Web Worm (Melissopus lateferreanus), Filbert Leaf Roller (Archips) Rosanus, Fruit Tree Leaf Roller (Archips)Argyrospiria, Grapeberry Moss (Paralobesia viteana), Grape Leaf Roller (Platynota sultana), Grape Leaf Skeletonizer (Harrisina americana (ground only)), Green Clover Worm (Plathypena scabra), Green Striped Maple Worm (Dryocampa rubicunda, Gummosos-Batrachedra, Comosae (Hodges)), Gypsy Moss (Lymantria dispar), Hemlock Trooper (Lambdina fiscellaria), Horn Worm (Manduca spp.), Imported Cabbage Worm (Pieris rapae, Io Moss (Automeris io), Jack Pine Bud Worm (Choristoneura pinus), Light Brown Apple Moss (Epiphyas postvittana), Melon Worm (Diaphania hyalinata), Mimosa Web Worm (Homadaula anisocentra), Oblique Banded Leaf Roller (Choristoneura rosaceana), Oleander Moss (Syntomeda epilais), Omnibara Three Flora (Playnota sultana), Omnibara Looper (Sabulodes aegrotata), Orange Dog (Papilio) Cresphontes, Orange Tortoise (Argyrotaenia citrana), Oriental Fruit Moss (Grapholita molesta), Peach Twig Boar (Anarsia lineatella), Pine Butterfly (Neophasia menapia), Podworm (Heliocoverpa zea), Red Banded Leaf Roller (Argyrotaenia velutinana), Red Humpt Caterpillar (Schizura concinna), Lindworm Complex (Various Leps.), Saddleback Caterpillar (Sibine stimulea), Saddle Prominent Caterpillar (Heterocampa) Guttivita, Salt Marsh Caterpillar (Estigmene)Acrea), Sodbed Worm (Crambus sp.), Span Worm (Ennomos subsignaria), Fall Canker Worm (Alsophylla pometaria), Spruce Bud Worm (Choristoneura fumiferana), Tent Caterpillar (Various Lasiocampidae), Thecla-Thecla Basilides (Geyr) Thecla basilides, Tobacco Horn Worm (Manduca sexta), Tobacco Moss (Ephestia elutella), Tufted Apple Bud Moss (Platynota idaeusalis), Twigboar (Anarsia) Lineatella, Bear-gated Cutworm (Peridroma saucia), Bear-gated Leaf Roller (Platynota flavedana), Velvet Bean Caterpillar (Anticarsia gemmatalis), Walnut Caterpillar (Datana intergerrima), Webworm (Hyphantria cunea), Western Tussock Moss (Orgyia vetusta), Southern Corn Stokeboar (Diatraea) Crambidoides, Corn Earworm, Sweet Potato Weevil, Pepper Weevil, Citrus Root Weevil, Strawberry Weevil, Pecan Weevil, Filbert Weevil, Ricewater Weevil, Alfalfa Weevil, Clover Weevil, Tee Shot Hole Bohr, Root Weevil, Sugarcane Beetle, Coffee Berry Bohr, Annual Bluegrass Weevil (Listronotus maculicollis), Asiatic Garden Beetle (Maladera castanea), European Chafer (Rhizotroqus majalis), Green June Beetle (Cotinis nitida), Japanese Beetle (Popillia japonica), May or June Beetle (Phyllophaga) sp.), Northern Masked Chafer (Cyclocephala borealis), Oriental Beetle (Anomala)orientalis), Japanese masked fly (Cyclocephala lurida), field beetle (Curculionoidea), Aedes aegypti, Busseola fusca, Chilo suppressalis, Culex pipiens, Culex quinquefasciatus, Diabrotica virgifera, Diatraea saccharalis, Helicoverpa armigera, Helicoverpa zea, Heliothis virescens, Leptinotarsa ​​decemlineata, Ostrinia furnacalis, Ostrinia nubilalis, Pectinophora gossypiella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Trichoplusia ni, and Xanthogaleruca Luteola.

106. The method according to claim 105, wherein the pest is selected from the group consisting of: Aedes aegypti, Busseoola fusca, Chilo suppressalis, Culex pipiens, Culex qinquefasciatus, Diabrotica virgifera, Diatraea saccharalis, Helicoverpa armigera, Helicoverpa zea, Heliothys virescens, Leptinotarsa ​​decemlineata, Ostrinia furnacalis, Ostrinia nubilalis, Pectinophora gossypiella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Trichoplusia ni, and Xanthogaleruca luteola.

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