Insecticide combinations

Abstract

New insecticidal combinations, compositions, and methods of using them are provided. The present invention is directed to combinations of cysteine-rich insecticidal peptides (CRIPs) and insecticides (IAs). The present invention also describes the use of such combinations and compositions to control insects. Described herein are genes encoding CRIPs, compositions and combinations comprising CRIPs and IAs, and methods using them useful for controlling pests.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 019,219, filed May 1, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application was filed on April 28, 2021 at 10:07 PM, and the Sequence Listing filed electronically herewith, entitled "225312-491452_ST25.txt" (1.41 MB), is incorporated by reference in its entirety.

[0003] Novel insecticidal combinations of cysteine-rich insecticidal proteins (CRIPs) and insecticides (IA), 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 said prokaryotes or eukaryotes), for the control and / or eradication of insect pests are described and claimed. [Background technology]

[0004] Many insects are vectors of disease. Anopheles mosquitoes are the primary vectors of Zika virus, chikungunya virus, and malaria, a disease caused by protozoan parasites of the genus Trypanosoma. Aedes aegypti is the primary vector for 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 Culex, Mansonia, and Anopheles mosquitoes.

[0005] Horse flies and deer flies can transmit the bacterial pathogens of tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as the parasitic roundworm (Loa loa) that causes loiasis in tropical Africa.

[0006] Eye gnats of the genus Hippelates can carry the spirochete pathogen that causes yaws (Treponema pertenue) and may also spread conjunctivitis (pink eye). Tsetse flies of the genus Glossina transmit the protozoan pathogens (Trypanosoma gambiense and T. rhodesiense) that cause African sleeping sickness. Sand flies of the genus Phlebotomus are vectors for the bacterium (Bartonella bacilliformis) that causes carrion disease (Oroyo fever) in South America. In parts of Asia and North Africa, they transmit the viral pathogen that causes sand fly fever (Papatati fever) as well as the protozoan pathogens (Leishmania spp.) that cause leishmaniasis.

[0007] Therefore, effective pesticide treatments are needed to preserve the crops we depend on for food and to protect human and animal health.

[0008] Described herein are combinations of insecticides (IA) and cysteine-rich insecticidal peptides (CRIPs). An IA can be one or more chemicals, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxic substances, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes and / or products derived therefrom (e.g., bacterial toxins), or eukaryotes and / or products derived therefrom (e.g., fungal toxins). The combination of IAs can provide an insecticidal effect greater than the additive effect of any of the IAs used in isolation.

[0009] In some embodiments, CRIPs are peptides, polypeptides, and / or proteins that have cysteine ​​residues capable of forming disulfide bonds; these disulfide bonds create a scaffolding motif observed in a wide variety of unrelated protein families. An example of a peptide within the CRIP family is the inhibitor cystine knot (ICK) peptide. ICK peptides include many molecules with insecticidal activity. Such ICK peptides are often toxic to naturally occurring biological target species (usually certain insects or arachnids). In many cases, ICK peptides may have arthropod origins, such as scorpion or spider venom.

[0010] Described herein are novel insecticidal combinations of IA and CRIP. For example, the present invention describes, inter alia, insecticidally effective combinations including (1) one or more CRIPs 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 the crops we depend on 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 Summary of the Invention [Means for solving the problem]

[0012] The present invention describes methods of combining CRIP and IA to provide insecticidal effects that are greater than the additive insecticidal effects of any IA or CRIP used alone. The present disclosure describes methods of making and using combinations of CRIP and IA to kill and control insects, even insecticide-resistant insects, at low doses. Without being bound by theory, our understanding of CRIP and IA will teach those skilled in the art and enable them to create new methods, compositions, compounds (proteins and peptides), and procedures for protecting plants and controlling insects.

[0013] The present disclosure describes a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA).

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

[0015] Additionally, the present disclosure describes compositions comprising a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), further comprising an excipient.

[0016] Additionally, the present disclosure describes a combination 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 according to the amino acid sequence set forth in SEQ ID NO:1.

[0017] Additionally, the present disclosure describes a combination 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 according to the amino acid sequence set forth in SEQ ID NO:2.

[0018] Additionally, the present disclosure describes a combination 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 according to the amino acid sequence set forth in SEQ ID NO:67.

[0019] Additionally, the present disclosure describes a combination 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 according to the amino acid sequence set forth in SEQ ID NO:65.

[0020] Additionally, the present disclosure describes a combination comprising Beauveria bassiana strain ANT-03 spores and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO:61.

[0021] Additionally, the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. tenebrionis strain NB-176 and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO:61.

[0022] Additionally, the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19 and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO:61.

[0023] Additionally, the present disclosure describes a combination comprising one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. israelensis strain BMP144 and a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO:61.

[0024] Additionally, the present disclosure describes a combination comprising a 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] Additionally, the present disclosure describes a combination comprising Galanthus nivalis agglutinin (GNA) and ACTX, wherein GNA has the amino acid sequence set forth in SEQ ID NO: 35, and the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.

[0026] Additionally, the present disclosure describes a combination comprising azadirachtin and ACTX, wherein azadirachtin has the chemical formula: 35 H 44 O 16 and ACTX is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.

[0027] Additionally, the present disclosure describes a combination comprising a boric acid compound and ACTX, wherein the boric acid compound has a chemical formula of H3BO3 and the ACTX peptide is a U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.

[0028] Additionally, the present disclosure describes a combination comprising Cydia pomonella granulovirus (CpGV) and ACTX, wherein the CpGV is Cydia pomonella granulovirus isolate V22 virus and the ACTX peptide is U+2-ACTX-Hv1a toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 61.

[0029] Furthermore, the present disclosure describes a method for controlling insects using a combination comprising a cysteine-rich insecticidal 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 insecticidal peptide (CRIP) and the insecticide (IA) to the site of the insect.

[0030] Furthermore, the present disclosure describes a method for controlling Bacillus thuringiensis toxin-resistant insects using a combination comprising 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 the site of the insect.

[0031] Additionally, the present disclosure describes a method of combating, controlling, or suppressing pests, comprising applying a pesticidally effective amount of a combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA) to the site of the pest or to a plant or animal susceptible to attack by the pest. [Brief explanation of the drawings]

[0032] [Figure 1]FIG. 1 shows a graph depicting 24-hour mortality of Aedes aegypti (mosquito) larvae following a diet incorporation assay using (1) U+2-ACTX-Hv1a with Bti, (2) Bti toxin alone, (3) U+2-ACTX-Hv1a alone, and (4) control (water). [Figure 2] A graph showing 3-day mortality of the Lepidopteran species beet armyworm (Spodoptera exigua) after a foliar spray assay using Bacillus thuringiensis var. kurstaki toxin (Btk) in combination with Γ-CNTX-Pn1a, where 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] A graph showing 3-day mortality of the Lepidopteran species, beet armyworm (Spodoptera exigua), following a foliar spray assay combining Btk and Av3-variant polypeptide (AVP), where (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 assessing WT-Ta1b degradation in Helicoverpa zea gut extract (HGE), a simulated lepidopteran gut environment, are shown after 0, 20, 40, 60, 180, and 1260 minutes. Boxes indicate the major and minor peaks, and thus, the degradation of WT-Ta1b. Nested insets show zoomed-in / zoomed views of the chromatograms. Boxes highlight peaks indicative of proteolytic events, evidenced by the presence of two shoulders; a smaller "shoulder" to the right of the major peak demonstrates a partial proteolytic event. [Figure 5]Chromatograms assessing TVP-R9Q degradation in Helicoverpa zea intestinal extract (HGE), a simulated Lepidopteran intestinal environment, are shown after 0, 20, 40, 60, 180, and 1260 minutes. Boxes indicate the presence of a single peak and therefore the stability of TVP-R9Q. Nested insets show zoomed-in / zoomed views of the chromatograms, where the presence of a single major peak (indicated by a box) indicates the stability of the TVP-R9Q peptide. [Figure 6] Graph showing the results of a defoliation assay when WT-Talb, Btk toxin, and their combination were tested against the lepidopteran species Helicoverpa zea (corn earworm). Treatments were as follows: (1) WT-Talb alone, (2) Btk toxin alone, (3) a combination of both WT-Talb and Btk toxin, or (4) a control (0.125% Vintre, a surfactant). Here, Btk toxin is indicated as "Btk." [Figure 7]

[0033] Figure 1 shows a graph depicting the results of a defoliation assay when TVP-R9Q, Btk toxin, and their combination were tested against the lepidopteran species Helicoverpa zea (corn earworm). Treatments were as follows: (1) TVP-R9Q alone, (2) Btk toxin alone, (3) a combination of both TVP-R9Q and Btk toxin, or (4) a control (0.125% Vintre, a surfactant). Here, Btk toxin is indicated as "Btk." [Figure 8] Graph showing the results of a mortality assay when WT-Talb, Btk toxin, and their combination were tested against the lepidopteran species Helicoverpa zea (corn earworm). Treatments were as follows: (1) WT-Talb alone, (2) Btk toxin alone, (3) a combination of both WT-Talb and Btk toxins, or (4) a control (0.125% Vintre, a surfactant). Here, Btk toxin is indicated as "Btk." [Figure 9]1 shows a graph depicting the results of a mortality assay when TVP-R9Q, Btk toxin, and their combination were tested against the lepidopteran species Helicoverpa zea (corn earworm). Treatments were as follows: (1) TVP-R9Q alone, (2) Btk toxin alone, (3) a combination of both TVP-R9Q and Btk toxin, or (4) a control (0.125% Vintre, a surfactant). Here, Btk toxin is indicated as "Btk." [Figure 10] FIG. 1 shows a graph depicting 4-day mortality of Coleoptera species darkling beetles (Mealworms) (Alphitobius diaperinus) after a diet incorporation assay with (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] 1 shows a graph depicting four-day mortality of Colorado potato beetles (Leptinotarsa ​​decemlineata) when sprayed with (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 12] 1 shows a graph depicting day 4 mortality in corn earworm larvae treated with: (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), where % w / v is the percent w / v of the total volume of the composition and the remainder is water. [Figure 13]1 shows a graph depicting mortality in newly hatched corn earworm neonates at day 3 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), where % w / v is percent w / v of the total volume of the composition, and the remainder is water. Proportional mortality refers to the proportion of individual insects that die over the course of 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% w / v). 1 shows a graph showing mortality of Fall armyworm (Spodoptera frugiperda) larvae 3 days after treatment with % w / v of the total composition, where % w / v is percent w / v of the total volume of the composition, the remainder being water. [Figure 15] 1 shows the chemical structure of the insect growth regulator azadirachtin. [Figure 16]Graphs showing mortality of corn earworms (Helicoverpa zea) on day 3 after treatment with (a) 0 μL / L azadirachtin (0% v / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control), (b) 80 μL / L azadirachtin (0.008% v / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v), (c) 0 μL / L azadirachtin (0% v / v), 10 mg / mL U+2-ACTX-Hv1a (1% w / v), and (d) 80 μL / L azadirachtin (0.008% v / v), 10 mg / mL U+2-ACTX-Hv1a (1% w / v). where % w / v is the percent w / v of the total volume of the composition, the remainder being water. [Figure 17] Graphs showing mortality of lesser mealworm (Alphitobius diaperinus) hatched larvae on day 3 after treatment with: (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). where % w / v is the percent w / v of the total volume of the composition, the remainder being water. [Figure 18]Graphs showing mortality in newly hatched Codling Moths (Cydia pomonella) larvae 7 days after treatment with: (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), where % w / v is the percent w / v of the total volume of the composition, the remainder being water. [Figure 19] (a) 0 μL / L CpGV (0% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v) (control), (b) 58.5 μL / L CpGV (0.00585% w / v), 0 mg / mL U+2-ACTX-Hv1a (0% w / v), (c) 0 μL / L CpGV (0% w / v), 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v), and (d) 58.5 μL / L CpGV (0.00585% w / v), 2 mg / mL U+2-ACTX-Hv1a (0.2% w / v) (where % 1 shows a graph showing mortality in hatched larvae of codling moth (Cydia pomonella) two days after treatment with 100 mg of ... [Figure 20]This graph shows the results of a feed incorporation assay of Novaluron and U+2-ACTX-Hv1a, assessing mortality in corn earworms (Helicoverpa zea) after 3 days. As shown, there was no evidence of a greater than additive effect when Novaluron and U+2-ACTX-Hv1a were combined in the corn earworm (Helicoverpa zea) feed incorporation 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] This graph shows the results of a diet incorporation assay of nanoparticles and U+2-ACTX-Hv1a, assessing mortality in corn earworms (Helicoverpa zea) after 3 days. As shown, there was no evidence of a greater than additive effect when nanoparticles and U+2-ACTX-Hv1a were combined in the corn earworm (Helicoverpa zea) diet incorporation 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 = number of dead insects divided by total number of insects. "UTC" means untreated control (water). [Figure 22]Figure 1 shows a graph depicting the mortality dose-response of a feed incorporation assay of cryolite and U+2-ACTX-Hv1a on corn earworms (Helicoverpa zea) after 3 days. As shown, there was no evidence of a greater-than-additive effect when combining cryolite and U+2-ACTX-Hv1a in a corn earworm (Helicoverpa zea) feed incorporation assay. Here, "U+2" refers to U+2-ACTX-Hv1a. The nanoparticle concentrations were as follows: (a) 10,000 ppm, (b) 2,000 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 = number of dead insects divided by the total number of insects. DETAILED DESCRIPTION OF THE INVENTION

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

[0034] "5' and 3' homology arms" or "5' and 3' arms" or "left and right arms" refer to polynucleotide sequences in a vector and / or targeting vector that homologously recombine with a target genomic sequence and / or endogenous gene of interest in a host organism to achieve successful genetic modification of a chromosomal locus in the host organism.

[0035] "Γ-CNTX-Pn1a" or "γ-CNTX-Pn1a" or "gamma-CNTX-Pn1a" or "gamma" refers to an insecticidal neurotoxin derived from the Brazilian armoured spider Phoneutria nigriventer. Γ-CNTX-Pn1a targets ionotropic glutamate receptors (GRINs) of the N-methyl-D-aspartate (NMDA) subtype 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 family Atracinae. ω / κ-HXTX-Hv1a is a positive allosteric modulator of nicotinic acetylcholine receptors and also mediates insect voltage-gated Ca2+ receptor activation. 2+ Channels and voltage-dependent K + The peptide may be a dual antagonist for 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, the disclosures of which are incorporated herein by reference in their entireties.

[0037] "ACTX" or "ACTX peptide" or "atracotoxin" 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] "ADN1 promoter" refers to a DNA segment consisting of the promoter sequence derived from the Schizosaccharomyces pombe adhesion defective protein 1 gene.

[0039] "Alpha-MF signal" or "αMF secretory signal" refers to a protein that directs a nascent recombinant polypeptide into the secretory pathway.

[0040] "Agriculturally acceptable carrier" includes all adjuvants, inert ingredients, dispersants, surfactants, adhesives, binders, etc. commonly used in pesticide formulation technology and are well known to those skilled in the art of pesticide formulation.

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

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

[0043] "Alignment" refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, 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, alignments can also be performed manually. Alignment programs typically iterate through potential alignments of the sequences, score the alignments using substitution tables, and use various strategies to arrive at the optimal potential 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 implementing one or more of the aforementioned algorithms 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 "phase shifts" and / or "gaps" into one or both of the sequences being compared to maximize the similarity between the two sequences, and scoring refers to a process that quantitatively expresses the relatedness of aligned sequences.

[0044] "Alpha-MF signal" or "αMF secretory signal" refers to a protein that directs a nascent recombinant polypeptide into the secretory pathway.

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

[0046] "Av2" or "ATX-II" or "Neurotoxin 2" or "Anemonia viridis toxin 2" or "δ-AITX-Avd1c" refers to a toxin isolated from the venom of Anemonia sulcata. An example of an Av2 polypeptide is the polypeptide having 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 alpha subunit III of the voltage-gated sodium channel. An example of an Av3 polypeptide is the Av3 polypeptide having the amino acid sequence of SEQ ID NO: 44 (NCBI Accession No. 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 non-naturally occurring polypeptide and / or polynucleotide sequence.

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

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

[0051] By "binary vector" or "binary expression vector" is meant an expression vector that can replicate itself in both E. coli and Agrobacterium strains, and that contains a region of DNA (often referred to as t-DNA) bracketed by left and right border sequences recognized by virulence genes, so that it can be copied by Agrobacterium and delivered into plant cells.

[0052] "bp" or "base pair" refers to a molecule containing two chemical bases bonded together. For example, a DNA molecule consists of two helical strands, each with a backbone made of alternating deoxyribose and phosphate groups. Each deoxyribose is bound 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 sporulation, Bacillus thuringiensis produces crystal proteins (i.e., proteinaceous inclusion bodies) called delta-endotoxins, which have insecticidal activity. In some embodiments, the Bt toxin can be a crystal (Cry) protein, a cytolytic (Cyt) protein, a plant-insecticidal protein (Vips), or other toxins produced by Bacillus thuringiensis.

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

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

[0056] "cDNA" or "copy DNA" or "complementary DNA" refers to a molecule that is complementary to a molecule of RNA. In some embodiments, cDNA can be either single-stranded or double-stranded. In some embodiments, cDNA can 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 any nucleic acid that shares the arrangement of sequence elements found in a naturally occurring mature mRNA species, the sequence elements being exons and 3' and 5' non-coding regions. Typically, mRNA species have contiguous exons, with intervening introns removed by nuclear RNA splicing to generate a continuous open reading frame that encodes a protein. In some embodiments, "cDNA" refers to DNA that is complementary to and derived from an mRNA template.

[0057] "CEW" refers to Corn earworm (Helicoverpa armigera).

[0058] For "cleavable linker" see linker.

[0059] "Cloning" refers to the process and / or methodology involved in 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., a plasmid) and directing the replication of the recombined DNA, or "recombinant DNA," usually by transforming the recombinant DNA into a bacterial or yeast host.

[0060] By "chimeric gene" is meant a DNA sequence that encodes a gene derived from parts of more than one coding sequence to produce a new gene.

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

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

[0063] "Combination" refers to any association between two or more items. The association can be spatial, temporal, and / or can refer to the use of two or more items for a common purpose. For example, a combination can be any spatiotemporal association, mixture, or permutation of (1) one or more CRIPs or pharmaceutically acceptable salts thereof, CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) described herein, where (1) and (2) are used for the common purpose of controlling or eradicating pest insects such that the pest insects die, stop or slow their movement, stop or slow their feeding, stop or slow their growth, become disoriented (e.g., with respect to navigation, food location, sleep behavior, and / or mating), fail to pupate, prevent reproduction, and / or prevent the insects from producing offspring, and / or prevent the insects from producing fertile offspring.

[0064] Unless the context makes clear otherwise, the term "combination" can include the simultaneous, separate, or sequential administration, in any order, of (1) one or more CRIPs 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).

[0065] It will be understood that in some embodiments, (1) one or more CRIP or pharmaceutically acceptable salts thereof, CRIP-insecticide protein 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" when a pest, or a site to which the pest is exposed, or a site to be protected from the pest (e.g., a plant), is treated by simultaneous exposure to both (1) and (2). In some embodiments, each of (1) one or more CRIP or pharmaceutically acceptable salts thereof, CRIP-insecticide protein or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) can be administered sequentially or according to different schedules, and individual doses of the different agents need not be administered at the same time or in the same composition. Rather, as long as both (1) one or more CRIPs or pharmaceutically acceptable salts thereof, CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, and (2) one or more insecticides (IA) remain pesticidal (i.e., have insecticidal activity), they are considered to be administered "in combination."

[0066] In some embodiments, "combination" refers to the simultaneous administration of (1) one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or a combination thereof, with (2) one or more insecticides (IA).

[0067] In some embodiments, "combination" refers to the separate administration of (1) one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or a combination thereof, with (2) one or more insecticides (IA).

[0068] In yet other embodiments, "combination" refers to the sequential administration, in any order, of (1) one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or a combination thereof, with (2) one or more insecticides (IA).

[0069] In some embodiments of the present invention, i.e., when the administration of the combination is sequential or separate, the delay in administering the second component should not be such as to eliminate the beneficial effect of the overall combination (i.e., the combination of (1) one or more CRIPs or pharmaceutically acceptable salts thereof, one or more CRIP-insecticidal proteins or pharmaceutically acceptable salts thereof, or combinations thereof, with (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 of each component can 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-insecticidal 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-insecticidal 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, a combination can be a "mixture." As used herein, a "mixture" refers to a combination of two or more agents, for example, (1) one or more CRIPs 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 each other and (2) one or more insecticides (IA).

[0072] As understood by those skilled in the art, "complementary" refers to the topological compatibility or correspondence of the interacting surfaces of two polynucleotides. Thus, two sequences are "complementary" to each other 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 stringent hybridization conditions. Thus, a polynucleotide whose sequence is 5'-TATAC-3' is complementary to a polynucleotide whose sequence is 5'-GTATA-3'.

[0073] "Conditioned medium" means a cell culture medium that is used by cells and is enriched in cellular materials but is cell-free.

[0074] "Cone shell" or "cone snail" or "cone" refers to an organism belonging to the genus Conus, a predatory marine gastropod. For example, in some embodiments, the 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 cone snails that acts by disrupting neuronal communication. For example, in some embodiments, the conotoxin can be an α-, ω-, μ-, δ-, or κ-conotoxin. Briefly, α-conotoxins (and αA- and φ-conotoxins) target nicotinic ligand-gated channels, ω-conotoxins target voltage-gated calcium channels, μ-conotoxins target voltage-gated sodium channels, δ-conotoxins target voltage-gated sodium channels, and κ-conotoxins target voltage-gated potassium channels.

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

[0077] "CRIP" refers to a cysteine-rich insecticidal peptide. CRIPs are peptides rich in cysteine ​​residues and, in some embodiments, are operable to form disulfide bonds between such cysteine ​​residues. In some embodiments, CRIPs contain at least four, sometimes six, and sometimes eight cysteine ​​amino acids in a protein or peptide having at least 10 amino acids, and the cysteines form two, three, or four disulfide bonds. In some embodiments, disulfide bonds contribute to the folding, three-dimensional structure, and activity of the insecticidal peptide. 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, CRIPs may or may not contain an inhibitor cystine knot (ICK) motif. For example, in some embodiments, a CRIP having an ICK motif may be an ACTX peptide derived from a spider, while in other embodiments, a CRIP lacking an ICK motif, i.e., a non-ICK CRIP, may be a peptide such as Av2 or Av3 isolated from a sea anemone. Non-ICK CRIPs may have four to eight cysteines that form two to four disulfide bonds. These cysteine-cysteine ​​disulfide bond-stabilized toxic peptides (CRIPs) can have remarkable stability when exposed to the environment. Many CRIPs have been isolated from venomous animals, such as spiders, scorpions, snakes, and sea snails, and are toxic to insects.

[0078] A "CRIP construct" refers to the three-dimensional arrangement / orientation of peptides, polypeptides, and / or motifs of operably linked polypeptide segments (e.g., a CRIP-insecticidal protein). For example, a CRIP-expressing ORF may include one or more of CRIP, an endoplasmic reticulum signal peptide (ERSP), a linker peptide (L), a translational stabilizing 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-expressing ORF. For example, in some embodiments, a CRIP construct describes the orientation of one of the following CRIP-insecticidal proteins, including, but not limited to, 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) N -STA, ERSP-STA-L-CRIP, ERSP-STA-CRIP-L, ERSP-STA-L-(CRIP)N ,ERSP-(STA-L) N -CRIP, ERSP-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 (where N is an integer ranging from 1 to 200.) See also "structural motif."

[0079] A "CRIP ORF diagram" refers to the composition of one or more CRIP ORFs described in diagrammatic or formulaic form. For example, a "CRIP ORF diagram" may be described using acronyms or short-hand references to the DNA segments contained within the ORF. Thus, in one example, a "CRIP ORF diagram" may describe polynucleotide segments encoding ERSP, L, STA, and CRIP by illustrating the DNA segments in formulaic form as "ersp" (i.e., the polynucleotide sequence encoding the ERSP polypeptide), "linker" or "L" (i.e., the polynucleotide sequence encoding the LINKER polypeptide), "sta" (i.e., the polynucleotide sequence encoding the STA polypeptide), and "crip" (i.e., the 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 these DNA segments.

[0080] A "CRIP polynucleotide" refers to a polynucleotide or group of polynucleotides operable to express and / or encode an insecticidal protein, including one or more CRIP in addition to one or more non-CRIP polypeptides or proteins.

[0081] "CRIP-insecticide protein" refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement consisting of: (1) at least one CRIP, or two or more CRIPs, and (2) an additional peptide, polypeptide, or protein, wherein the additional peptide, polypeptide, or protein is capable of doing one or more of the following: (a) increasing mortality and / or inhibiting insect growth when insects are exposed to the CRIP-insecticide protein compared to CRIP alone, (b) increasing expression of the CRIP-insecticide protein, e.g., in a host cell or expression system, and / or (c) affecting post-translational processing of the CRIP-insecticide protein.

[0082] In some embodiments, an insecticidal protein can comprise one or more CRIPs disclosed herein. In some embodiments, a CRIP-insecticidal protein can be a polymer comprising two or more CRIPs. In some embodiments, an insecticidal protein can comprise a CRIP homopolymer (e.g., two or more CRIP monomers where the CRIP is the same). In some embodiments, an insecticidal protein can comprise a CRIP heteropolymer (e.g., two or more CRIP monomers where the CRIP monomers are different).

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

[0084] In some embodiments, a CRIP-insecticidal protein can be a polymer comprising two or more CRIPs, where the CRIPs are operably linked via a linker peptide (e.g., a cleavable and / or non-cleavable linker). In some embodiments, a CRIP-insecticidal protein can refer to one or more CRIPs operably linked to one or more proteins, such as a stabilization 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, a CRIP-insecticidal protein can be a non-naturally occurring protein comprising (1) a wild-type CRIP protein and (2) an additional peptide, polypeptide, or protein (e.g., an ERSP, a linker, a STA, a UBI, or a histidine tag or similar marker).

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

[0086] "Culturing" refers to the growth of organisms on or in various types of media. For example, the term "culturing" can refer to the growth of a population of cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to the fermentative recombinant production (typically in a vessel or reactor) of a heterologous polypeptide of interest and / or other desired end product.

[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] By "defined medium" is meant a medium that is composed of known chemical components but does not contain crude proteinaceous extracts or by-products such as yeast extract or peptones.

[0089] "Degeneracy" or "codon degeneracy" refers to the phenomenon in which one amino acid can be coded for by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule encoding a protein or polypeptide can vary due to degeneracy. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide having a particular activity, and such functionally equivalent variants are contemplated herein.

[0090] "Desmethylrimocin 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] "Disulfide bond" means a covalent bond between two cysteine ​​amino acids derived by the coupling of two thiol groups on the side chains.

[0092] "DNA" refers to deoxyribonucleic acid and includes 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- or double-stranded form. For example, one or more nucleotides form a polynucleotide.

[0093] "dNTP" refers to the nucleoside triphosphates that make up DNA and RNA.

[0094] A "dual expression cassette" refers to expression cassettes for two heterologous polypeptides contained on the same vector.

[0095] "Double transgene peptide expression vector" or "double transgene expression vector" means a yeast expression vector that contains two copies of a heterologous polypeptide expression cassette.

[0096] "Endogenous" refers to a naturally occurring and / or organism-present polynucleotide, peptide, polypeptide, protein, or process, e.g., a molecule or activity already present in a host cell prior to specific genetic manipulation.

[0097] An "enhancer element" refers to a DNA sequence operably linked to a promoter that is capable of exerting increased transcriptional activity on the promoter compared to the transcriptional activity that results from the promoter in the absence of the enhancer element.

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

[0099] "ERSP" or "endoplasmic reticulum signal peptide" is an N-terminal sequence of amino acids that is recognized and bound by the host cell's signal recognition particle during protein translation of an mRNA molecule encoding CRIP, which translocates the protein-translating ribosome / mRNA complex 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 a polynucleotide encoding 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] The term "excipient" refers to any pharmacologically inactive, natural, or synthetic ingredient or substance that is formulated with (e.g., simultaneously with) or subsequently with an active ingredient of the present invention (i.e., CRIP or a CRIP-insecticidal protein). In some embodiments, an excipient can be any additive, adjuvant, binder, filler, carrier, coating agent, diluent, disintegrant, filler, glidant, lubricant, preservative, vehicle, or combination thereof that can be administered with a CRIP or a CRIP-insecticidal protein of the present invention and / or is useful in preparing a composition of the present invention. Excipients include any such material known in the art that is non-toxic and does not interact with other components of the composition. In some embodiments, an excipient can be formulated with a CRIP or a CRIP-insecticidal protein when the composition is prepared for the purpose of increasing the composition's weight (and is therefore often referred to as a filler, bulking agent, or diluent). In other embodiments, an excipient can be used to enhance the active ingredient in the final dosage form, such as by promoting absorption and / or solubility. In still other embodiments, excipients can be used to provide stability or to prevent contamination (e.g., microbial contamination). In other embodiments, excipients can be used to impart physical characteristics to a composition (e.g., a composition that is in the physical form of a dry granule or a dry flowable powder). Reference to an excipient includes both one and more than one such excipient. Suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.

[0103] "Expression cassette" refers to (1) a DNA sequence of interest (e.g., a polynucleotide operable to encode CRIP) and one or more of the following: (2) a promoter, terminator, and / or enhancer elements, (3) an appropriate 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) through (6) is referred to as an "expression cassette." In some embodiments, there may be multiple expression cassettes cloned into a vector. For example, in some embodiments, there may be a first expression cassette comprising a polynucleotide operable to encode CRIP. In alternative embodiments, there are two expression cassettes (i.e., a dual expression cassette), each comprising a polynucleotide operable to encode CRIP. In other embodiments, there are three expression cassettes (i.e., a triple expression cassette) operable to encode CRIP. In some embodiments, a dual expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette. In some embodiments, a triple expression cassette can be generated by subcloning a third expression cassette into a vector comprising a first expression cassette and a second expression cassette. Methods relating to expression cassettes and cloning techniques are well known in the art and described herein. See also CRIP expression cassette.

[0104] "Expressed ORF" means the nucleotides encoding a protein complex and is defined as the nucleotides of an ORF.

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

[0106] "Fermentation beer" refers to spent fermentation medium (i.e., fermentation medium supernatant after removal of organisms) that has been inoculated into and consumed by transformed host cells (e.g., yeast cells operable to express a CRIP of the present invention). In some embodiments, fermented beer refers to the solution recovered after fermentation of transformed host cells. The term "fermentation" broadly refers to the enzymatic and anaerobic or aerobic breakdown of organic matter (e.g., carbon substrates) by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, etc.) to produce a fermentation product (e.g., one or more peptides of the present invention). Fermentation, as used herein, typically describes a process that occurs under anaerobic conditions; however, the term "fermentation" as used herein can also occur in processes that occur in the presence of oxygen, and therefore, it is not intended that the term be limited to strictly anaerobic conditions.

[0107] "Fermentation solids" refers to the solids (including dissolved solids) remaining from fermenting beer during the yeast-based fermentation process and consists essentially of salts, complex protein sources, vitamins, and additional yeast by-products with a molecular weight cutoff of about 200 kDa to about 1 kDa.

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

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

[0110] "Homologous" refers to sequence similarity or sequence identity between two polypeptide molecules or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of the two DNA molecules is occupied by adenine), the molecules are homologous at that position. The percent 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 compared positions, multiplied by 100. Thus, in some embodiments, the term "homologous" refers to sequence similarity between two polypeptide molecules or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of the two DNA molecules is occupied by adenine), the 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, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology.

[0111] When used in reference to nucleic acids, the term "homology" refers to the degree of complementarity. There can be partial or complete homology (and therefore identity). "Sequence identity" refers to a measure of relatedness between two or more nucleic acids and is given as a percentage with reference to the total length compared. Identity calculations take into account those nucleotide residues that are identical and in the same relative positions in each larger sequence.

[0112] "Homologous recombination" refers to the event of replacing a segment of DNA with another segment that has identical (homologous) or nearly similar regions. For example, in some embodiments, "homologous recombination" refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or two identical molecules of DNA. Briefly, 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. Homologous recombination varies greatly between different organisms and cell types, but most forms involve the same basic steps: after a double-strand break is created, a section of DNA surrounding the 5' end of the break is cut in a process called resection. Subsequently, in a strand invasion step, the overhanging 3' end of the cut DNA molecule then "invades" an uncut similar or identical DNA molecule. After strand invasion, the further sequence of events may follow either of two major 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 viruses, suggesting that it is a nearly universal biological mechanism. For example, in some embodiments, homologous recombination can occur using site-specific integration (SSI) sequences, whereby there is a strand exchange crossover event between nucleic acid sequences that are substantially similar in nucleotide composition. These crossover events can occur between a sequence contained in a targeting construct of the present invention (i.e., an SSI sequence) and an endogenous genomic nucleic acid sequence (e.g., a polynucleotide encoding a peptide subunit). Furthermore, in some embodiments, two or more site-specific homologous recombination events can occur, which will result in a replacement event in which a nucleic acid sequence contained in the targeting construct replaces a specific sequence present in the endogenous genomic sequence.

[0113] "ICK motif" or "ICK motif protein" or "inhibitor cystine-knot motif" or "ICK peptide" or "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 bridges, where the three disulfide bridges are covalent bonds, and the covalent disulfide bonds among the six half-cystine residues are between the first and fourth, second and fifth, and third and sixth half-cystines of the six half-cystine core amino acids starting from the N-terminal amino acid. Generally, this type of peptide contains a beta-hairpin secondary structure, usually composed of residues located between the fourth and sixth core half-cystines of the motif, where the hairpin is stabilized by the structural bridges 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 nucleotides encoding an ICK motif protein.

[0115] "ICK motif protein expression ORF" or "expression ORF" means the nucleotides encoding an ICK motif protein complex, defined as the nucleotides in the ORF.

[0116] "ICK motif protein expression vector" or "ICK expression vector" or "ICK motif expression vector" refers to a binary vector containing an expression ORF. The binary vector also contains the necessary transcription promoter and terminator sequences surrounding the expression ORF to facilitate expression of the ORF and the protein it encodes.

[0117] "Identity" refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The term "identity" also means the degree of sequence relatedness between polypeptide sequences or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by any one of numerous methods known to those skilled in the art, including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., 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 Mathematics, 1999. Math., 48:1073 (1988), the disclosures of which are incorporated herein by reference in their entireties. Moreover, methods to determine 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.F. 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, Md. 20894; Altschul, S., et al. J. Mol. Biol. 215:403-410(1990)), the disclosures of which are incorporated herein by reference in their entireties.

[0118] "IGER" refers to the name of a short peptide based on the actual single-letter code sequence. This is an example of an intervening linker.

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

[0120] "Inactive" refers to the state of something being out of use, e.g., dormant and / or non-functional. For example, when used in the context of or referring to a gene, the term inactive means that the gene no longer actively synthesizes a gene product, translates 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 failure of a gene to transcribe RNA, failure of RNA processing (e.g., pre-mRNA processing, RNA splicing, or other post-transcriptional modification), interference with non-coding RNA maturation, interference with RNA transport (e.g., from the nucleus to the cytoplasm), interference with translation, protein folding, translocation, protein transport, and / or inhibition and / or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that contribute to any of the foregoing processes.

[0121] "Inoperable" refers to a state of not functioning, malfunctioning, or no longer being able to function. For example, when used in the context of or referring to a gene, the term inoperable means that the gene is no longer able to operate 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, translate the gene product into a protein, or otherwise cannot perform its normal function. For example, in some embodiments, the term inoperable can refer to failure of a gene to transcribe RNA, failure of RNA processing (e.g., pre-mRNA processing, RNA splicing, or other post-transcriptional modification), interference with non-coding RNA maturation, interference with RNA transport (e.g., from the nucleus to the cytoplasm), interference with translation, protein folding, translocation, protein transport, and / or inhibition and / or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that contribute to any of the foregoing processes.

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

[0123] "Insect gut environment" or "gut environment" refers to the specific pH and proteinase conditions found within the foregut, midgut, or hindgut of an insect or insect larva.

[0124] By "insect hemolymph environment" is meant the specific pH and proteinase conditions found within an insect or insect larva.

[0125] "Insecticidal activity" refers to the ability of an insect to die, stop or slow its movement, stop or slow its feeding, stop or slow its growth, become disorganized (e.g., with respect to navigation, food location, sleep behavior, and / or mating), fail to pupate, prevent reproduction, and / or prevent the insect from producing offspring, and / or prevent the insect from producing fertile offspring, upon or after exposure of the insect to the compound, substance, or peptide.

[0126] "Insecticide" or "IA" or "agent" refers to one or more chemicals, 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 agents produced by such prokaryotes or eukaryotes). In some embodiments, IAs include, but are not limited to, RNAi, digestive poisons, type 0 chitin biosynthesis inhibitors, type 1 chitin biosynthesis inhibitors, insect viruses, Azadirachta indica, compounds with unknown MOA, bacteria (and products thereof), fungi (and products thereof), nematodes (and products thereof), plant extracts, mechanical disruption agents, optical brighteners, silica nanospheres, chitinases, lectins, membrane attack complex / perforin (MACPF) proteins, plant virus coat protein-toxin fusions, glycan-binding domain / toxin fusion proteins, acetylcholinesterase (AchE) inhibitors, GABA-gated chloride channel blockers, sodium channel modulators, nicotinic acetylcholine receptor (nAchR) competitive modulators, nicotinic acetylcholine receptor (nAchR) allosteric modulators - site I, glutamate-gated chloride channel (GluCl) allosteric modulators, juvenile hormone mimetics, and other non-specific compounds. and GABA-gated chloride channel allosteric modulators, inhibitors of acetyl-CoA carboxylase, mitochondrial complex IV electron transport inhibitors, mitochondrial complex II electron transport inhibitors, ryanodine receptor modulators, chordotonal organ modulators - undefined target site, or GABA-gated 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 made and / or used according to any of the peptide and / or protein methods described herein.

[0127] By "integrating expression vector" or "integrating vector" is meant a yeast expression vector that can insert itself into a specific locus in the yeast cell genome and become a stable part of the yeast genome.

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

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

[0130] "Isolated" refers to separating an entity and / or component from its natural environment, for example, a toxin isolated from a given genus or species means that the toxin is separated from its natural environment (e.g., removed from the wild-type organism).

[0131] "Kappa-ACTX peptide" refers to an excitotoxin that inhibits insect calcium-activated potassium (KCa) channels (Slo type). As used herein, "kappa-ACTX peptide" may refer to a peptide isolated from the Australian Blue Mountains funnel-web spider, Hadronyche versuta, or variants thereof.

[0132] "kb" refers to kilobase (i.e., 1000 bases). As used herein, the term "kb" refers to the length of a nucleic acid molecule. For example, 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long. A 1 kb-long double-stranded DNA molecule contains 2,000 nucleotides (i.e., 1,000 nucleotides on each strand). Alternatively, a 1 kb-long single-stranded RNA molecule contains 1,000 nucleotides.

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

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

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

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

[0137] "1" or "linker" refers to the nucleotides encoding the linker peptide.

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

[0139] "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 strong, inducible reporter gene that can be used to drive the expression of exogenous genes transformed into yeast.

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

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

[0142] "LD 50 " refers to the lethal dose of 50, which means the dose required to kill 50% of a population.

[0143] "Lepidopteran gut environment" refers to the particular pH and proteinase conditions found within the foregut, midgut, or hindgut of a Lepidopteran insect or larva.

[0144] "Lepidopteran hemolymph environment" refers to the specific pH and proteinase conditions found within Lepidopteran insects or larvae.

[0145] "Linker" or "LINKER" or "peptide linker" or "L" or "intervening linker" refers to a short peptide sequence operable 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 independent secondary and tertiary structure formation during translation. In some embodiments, the linker may be either resistant or susceptible to cleavage in the plant cell environment, the insect and / or Lepidopteran gut environment, and / or the insect hemolymph and Lepidopteran hemolymph environment. In some embodiments, the linker may be cleavable by a protease. For example, in some embodiments, the linker can be cleaved by a plant protease (e.g., papain, bromelain, ficin, actinidin, zingibain, and / or cardosin), an insect protease, a fungal protease, a vertebrate protease, an invertebrate protease, a bacterial protease, a mammalian protease, a reptilian protease, or an avian protease. In some embodiments, the linker can be cleavable or non-cleavable. In some embodiments, the linker comprises a secondary or tertiary region, each region cleavable by at least two types of proteases, one of which is an insect and / or nematode protease and the other of which is a human protease. In some embodiments, the linker can have one of (at least) three roles: cleaved in the insect gut environment, cleaved in plant cells, or intentionally designed to not be cleaved.

[0146] "Medium" (plural "media") refers to a nutrient solution for growing cells in cell culture.

[0147] "MOA" refers to 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: a sample of interest is separated on a gel using a set of molecular weight standards. The sample is run, and then the gel is treated with the desired stain, followed by destaining for approximately 2-14 hours. The next step is to determine the relative migration distance (Rf) of the standards and the protein of interest. The migration distance can be determined using the following formula:

number

[0149] The logarithm of the MW can then be determined based on the values ​​obtained for the bands in the standards, e.g., in some embodiments, the logarithm of the molecular weight of the SDS-denatured polypeptide and its relative migration distance (Rf) are plotted on a graph. After plotting the graph, interpolation of the derived values ​​will yield the molecular weight of the unknown protein band.

[0150] "Motif" refers to a polynucleotide or polypeptide sequence that is associated with some biological significance and / or exerts some effect or is involved in some biological process.

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

[0152] A "variant" refers to an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein that has a change or variation (e.g., in a nucleotide sequence or amino acid sequence) that causes the organism and / or sequence to differ from a naturally occurring or wild-type organism, wild-type sequence, and / or reference sequence to which the variant is compared. In some embodiments, the change or variation can be one or more nucleotide and / or amino acid substitutions or modifications (e.g., deletions or additions). In some embodiments, the one or more amino acid substitutions or modifications can be conservative, where such conservative amino acid substitutions and / or modifications in a "variant" do not substantially reduce the activity of the variant relative to the non-mutated form of the variant. For example, in some embodiments, a "variant" has one or more conservative amino acid substitutions when compared to a peptide having a disclosed and / or claimed sequence, as indicated by a SEQ ID NO:

[0153] "N-terminus" refers to the free amine group (ie, -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 a peptide having 4-8 cysteines that form 2-4 disulfide bonds. Non-ICK peptides include cystine knot peptides that are not ICK peptides. Non-ICK peptides may have a disulfide bond pattern that differs from ICK. Examples of non-ICK CRIPs are peptides such as Av2 and Av3 isolated from sea anemones. These sea anemone peptides are examples of a class of compounds that modulate sodium channels in the insect peripheral nervous system (PNS).

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

[0158] "Normalized peptide yield" refers to the peptide yield in a conditioned medium divided by the corresponding cell density at the time the peptide yield is 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 an HPLC chromatograph (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 because the 600 nm wavelength prevents cells from being damaged by excessive UV light.

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

[0161] "Omega peptide" or "omega toxin" or "omega-ACTX-Hv1a" or "native omega-ACTX-Hv1a" all refer to an ACTX peptide originally isolated from the Australian Blue Mountains funnel-web spider, known as Hadronyche versuta. The omega peptide is a positive allosteric modulator of nicotinic acetylcholine receptors and also a voltage-gated Ca receptor modulator in insects. 2+ Channels and voltage-dependent K +

[0013] The present invention may be a dual antagonist for 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, the disclosures of which are incorporated herein by reference in their entireties.

[0162] "One-letter code" refers to a peptide sequence in which the one-letter code is listed to distinguish the 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" refers to the ability to be used, to do something, and / or to achieve some function or result. For example, in some embodiments, "operable" refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and / or protein. For example, in some embodiments, a polynucleotide may be operable to encode a protein, meaning that the polynucleotide contains information embedded in it with the ability to produce a protein (e.g., by transcribing mRNA that is subsequently translated into a protein).

[0164] "Operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting them to function in their intended manner. For example, in some embodiments, operably linked can refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operably linked can mean that two adjacent DNA sequences are positioned together so that the transcriptional activation of one DNA sequence can affect the other DNA sequence. In still other embodiments, the term "operably linked" can refer to two or more peptides and / or polypeptides that are linked in such a way as to produce a single polypeptide chain, or alternatively, the term operably linked can refer to two or more peptides that are linked in such a way that one peptide has some effect on the other. In still other embodiments, operably linked DNA sequences can refer to two adjacent DNA sequences that are positioned together so that the transcriptional activation of one can affect the other.

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

[0166] In some embodiments, an ORF is a continuous string of codons beginning with a start codon (usually ATG in DNA and AUG in RNA) and ending with a stop codon (usually UAA, UAG, or UGA). In other embodiments, an ORF can be a stretch of RNA or DNA sequence between a translation initiation signal (e.g., AUG or ATG) and any one or more known stop codons, where the stretch of RNA or DNA sequence encodes one or more polypeptide sequences. In some other embodiments, an ORF can be a DNA sequence that encodes a protein beginning with an ATG start codon and ending with a TGA, TAA, or TAG stop codon. ORF can also refer to the translated protein that the DNA encodes. 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 contemplates a series of codons that does not include a stop codon. Thus, while an ORF may contain introns, a coding sequence is 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 ribosomal translation machinery (i.e., a coding sequence does 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) flanked by two site-specific recombination sites (e.g., 5' and 3' nucleotide sequences of a target gene that are homologous to homology arms of a targeting vector) during in vivo homologous recombination. See "knockout."

[0168] "Parasporal crystal toxin" refers to any peptide, polypeptide, and / 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 the parasporal body or parasporal crystal is ingested by an insect, the toxin-containing parasporal crystal dissolves in alkaline gut fluid and is then cleaved by protoxin midgut proteases to generate the active peptide toxin (e.g., δ-endotoxin).

[0169] By "peptide expression cassette" or "expression cassette" is meant a DNA sequence that is comprised of all the DNA elements necessary to complete transcription of an insecticidal protein in a biological expression system. In the methods described herein, this includes a transcription promoter, a DNA sequence encoding an α-mating factor signal sequence, a cleavage site, an insecticidal protein transgene, a stop codon, and a transcription terminator.

[0170] By "peptide expression vector" is meant an expression vector in a host organism that contains a heterologous peptide transgene.

[0171] By "peptide-expressing yeast strain", "peptide-expressing strain" or "peptide-producing strain" is meant a yeast strain capable of producing a heterologous peptide.

[0172] "Peptide-IA" refers to an insecticide that is an amino acid, peptide, polypeptide, and / or protein.

[0173] "Peptide transgene" or "insecticidal peptide transgene" or "insecticidal protein transgene" refers to a DNA sequence that encodes a peptide of interest and is capable of being translated in a biological expression system.

[0174] "Peptide yield" refers to the concentration of insecticidal peptide in conditioned medium produced by cells of a peptide-expressing yeast strain, which 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 an HPLC chromatograph (e.g., mAu.sec).

[0175] "Peritrotrophic membrane" refers to the surface layer in the insect gut that protects the gut wall and can aid in the movement of large food particles through the gut while still allowing digestion.

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

[0177] A "pesticidally effective amount" refers to an amount of a pesticide capable of causing death to at least one pest or significantly reducing the growth, feeding, or normal physiological development of the pest. This amount will vary depending on factors such as, for example, the particular target pest to be controlled, the particular environment, location, plant, crop, or agricultural land to be treated, environmental conditions, and the method, rate, concentration, stability, and amount of application of the pesticidally effective polypeptide composition. Formulations may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of pest infestation.

[0178] "Pharmaceutically acceptable salt" is synonymous with agriculturally acceptable salt and, as used herein, refers to a compound that has been modified by making an acid or base salt thereof.

[0179] "Plant" is intended to mean whole plants, plant tissues, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, and their progeny. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0180] By "plant transgenic protein" is meant 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] By "plant-cleavable linker" is meant a cleavable linker peptide, or nucleotides encoding a cleavable linker peptide, that contains a plant protease recognition site and can be cleaved during the protein expression process in a plant cell.

[0182] "Plant-incorporated protectant" or "PIP" means an insecticidal protein produced by a transgenic plant and the genetic material necessary for the plant to produce that protein.

[0183] "Plasmid" refers to a DNA segment that functions as a carrier of a gene of interest and, when transformed or transfected into an organism, is capable of replicating and expressing the DNA sequence contained therein 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 DNA fragments and / or to select, through some selection, a population of hosts that carry the plasmid) or an "expression plasmid" (i.e., a plasmid used to produce large amounts of polynucleotides and / or polypeptides).

[0184] A "polar amino acid" is an amino acid that is polar and includes serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan, and tyrosine, with preferred polar amino acids being serine, threonine, cysteine, asparagine, and glutamine, and serine being most preferred.

[0185] "Polynucleotide" refers to a polymeric form of nucleotides of any length (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) (e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides). As used herein, the term "polynucleotide" includes double- and single-stranded DNA, as well as double- and single-stranded RNA, and also includes modified and unmodified forms of polynucleotides (modifications to and of polynucleotides can include, for example, methylation, phosphorylation, and / or capping). In some embodiments, a polynucleotide can be a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, a cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, an isolated DNA of any sequence, an isolated RNA of any sequence, a nucleic acid probe, a primer, or an amplified copy of any of the foregoing.

[0186] In yet other embodiments, a polynucleotide can refer to a polymeric form of nucleotides that is operable to encode an open reading frame of a gene.

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

[0188] In some embodiments, polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The structure of a polynucleotide can be referenced by its 5' or 3' end, or ends, which indicate the orientation of the polynucleotide. Adjacent nucleotides in a single strand of a polynucleotide are typically linked by a phosphodiester bond between their 3' and 5' carbons. However, different nucleotide linkages, including methylene, phosphoramidate, and the like, can also be used. This means that the respective 5' and 3' carbons can be exposed at either end of a polynucleotide (which can be referred to as the 5' and 3' ends, or ends). The 5' and 3' ends can also be referred to as phosphoryl (PO4) and hydroxyl (OH) ends, due to the chemical groups attached to the respective ends. The term polynucleotide also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment making or using a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0189] In some embodiments, a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, including nucleotides with unnatural bases and nucleotides with modified natural bases, such as azapurines or deazapurines. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polynucleotide.

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

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

[0192] The term "RNA molecule" or ribonucleic acid molecule refers to a polynucleotide that has a ribose sugar, rather than a deoxyribose sugar, as one of its pyrimidine bases, and typically has uracil, rather than 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, RNA molecules can include single-stranded molecules transcribed from DNA in a cell nucleus, mitochondria, or chloroplast, which have a linear sequence of nucleotide bases complementary to the DNA strand from which they are transcribed.

[0193] In some embodiments, the polynucleotide can further comprise one or more heterologous regulatory elements. For example, in some embodiments, the regulatory element is one or more of a promoter, an enhancer, a silencer, an operator, a splicing signal, a polyadenylation signal, a termination signal, an RNA transport element, an internal ribosome entry site (IRES), a polyU sequence, or a combination thereof.

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

[0195] "Post-transcriptional regulatory elements" are DNA segments and / or mechanisms that affect an mRNA after it is transcribed. Post-transcriptional mechanisms include splicing events, capping, addition of poly(A) tails, and other mechanisms known to those of skill in the art.

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

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

[0198] "Ratio" refers to a quantitative relationship between two quantities that indicates the number of times one value is contained within or subsumed by the other value.

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

[0200] "Recombinant DNA" or "rDNA" refers to DNA that is composed of two or more distinct 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 aspect 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. Regulatory elements can be cis-regulatory elements (CREs) or trans-regulatory elements (TREs). In some embodiments, regulatory elements can be one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA transport elements, internal ribosome entry sites (IRESs), polyU sequences, and / or other elements that affect gene expression (e.g., increase or decrease expression in a tissue-specific, time-dependent manner, and / or cause constitutive expression).

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

[0204] A "restriction site" refers to a location 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, salanin is C 34 H 44 It has a molecular formula of O9 and a molecular weight of 596.7 g / mol.

[0206] "Sea anemones" refers to a group of marine animals in the order Actiniaria. Sea anemones are named after the terrestrial flowering plant sea anemone due to the colorful appearance of many sea anemones. For example, in certain embodiments, the 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] By "selection gene" is meant a gene that confers an advantage to a genetically modified organism for growth under selective pressure.

[0208] "Serovar" or "serotype" refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, serotypes are antigenically and serologically distinct varieties of microorganisms.

[0209] "sp." refers to 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 vector of interest. For example, a polynucleotide encoding a variant or peptide can be subcloned into a pKlac1 plasmid, followed by selection of yeast colonies transformed with the pKLAC1 plasmid.

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

[0213] "STA" or "translational stabilizing protein" or "stabilizing domain" or "stabilizing protein" (used interchangeably herein) refers to a peptide or protein with sufficient tertiary structure to accumulate intracellularly without being targeted by the cellular process of proteolysis. The protein can be 5 to 50 amino acids in length. The translational stabilizing protein is encoded by a DNA sequence of a protein operably linked to an ORF encoding an insecticidal protein or CRIP. The operably linked STA can be either upstream or downstream of CRIP, and can 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 translational stabilizing protein may also have activity to increase delivery of CRIP across the gut wall into the insect hemolymph.

[0214] "sta" refers to a nucleotide sequence that encodes a translation stabilizing protein.

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

[0216] "Talb" or "U1-agatoxin-Talb" or "TalbWT" or "wild-type U1-agatoxin-Talb" refers to a polypeptide isolated from the hobo spider Eratigena agrestis. An example of U1-agatoxin-Talb is the polypeptide having the amino acid sequence of SEQ ID NO: 1 (NCBI accession number O46167.1).

[0217] A "Talb variant polynucleotide" or "U1-agatoxin-Talb variant polynucleotide" refers to a polynucleotide or group of polynucleotides operable to express and / or encode an insecticidal protein, including one or more TVPs. The term "U1-agatoxin-Talb variant polynucleotide" when used to describe a U1-agatoxin-Talb variant polynucleotide sequence contained in a TVP expression ORF, when it is contained in a vector, and / or when describing a polynucleotide encoding an insecticidal protein, is described as "tvp" and / or "Tvp."

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

[0219] "Transfection" and "transformation" both refer to the process of introducing exogenous and / or heterologous DNA or RNA (e.g., a vector containing a polynucleotide encoding CRIP) into a host organism (e.g., a prokaryote or 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 may reserve the term "transfection" for the process of describing the introduction of exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms "transformation" and "transfection" are used interchangeably, regardless of whether the process describes the introduction of exogenous and / or heterologous DNA or RNA into prokaryotes (e.g., bacteria) or eukaryotes (e.g., yeast, plants, or animals).

[0220] "Transgene" means a heterologous DNA sequence encoding a protein that is transformed into a plant.

[0221] By "transgenic host cell" is meant a cell that has been transformed with a gene and selected for its transgenic state via an additional selection gene.

[0222] By "transgenic plant" is meant a plant derived from a single cell that has been transformed with foreign DNA such that all cells of the plant contain the transgene.

[0223] "Transient expression system" refers to an Agrobacterium tumefaciens-based system that delivers DNA encoding a disarmed plant virus into plant cells where it is expressed. The plant virus is engineered to express the protein of interest at high concentrations, up to 40% of the TSP.

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

[0225] "TRBO" refers to a transient plant expression system using tobacco mosaic virus with the viral coat protein gene deleted.

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

[0227] "TVP" or "U1-agatoxin-Talb variant polypeptide (TVP)" or "Talb variant polypeptide (TVP)" refers to a mutant or variant of a wild-type U1-agatoxin-Talb polypeptide sequence and / or a polynucleotide sequence encoding the wild-type U1-agatoxin-Talb polypeptide, which has been modified to produce a non-naturally occurring polypeptide and / or polynucleotide sequence. An exemplary wild-type U1-agatoxin-Talb polypeptide sequence having the amino acid sequence of SEQ ID NO: 1 is provided herein. An exemplary wild-type U1-agatoxin-Talb precursor polypeptide sequence is provided herein, having the amino acid sequence of SEQ ID NO: 48 (NCBI Accession No. O46167.1), including the signal sequence "MKLQLMICLVLLPCFFC" (SEQ ID NO: 59). In some embodiments, TVP can have an amino acid sequence according to any of the amino acid sequences listed in Table 1. Accordingly, the term "TVP" refers to a peptide having one or more mutations compared to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TVP can have an amino acid sequence according to formula (I). EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 Formula (I)

[0228] 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 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, the TVP can have an amino acid sequence according to formula (II), or a pharmaceutically acceptable salt thereof. EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG Formula (II)

[0230] 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 is R or Q, and Z1 is T or A.

[0231] "U-ACTX-Hv1a" or "hybrid peptide" or "hybrid toxin" or "hybrid-ACTX-Hv1a" or "native hybrid ACTX-Hv1a" or "U peptide" or "U toxin" or "native U" or "native U-ACTX-Hv1a" all refer to the ACTX peptide, which was discovered in the Australian Blue Mountains funnel-web spider, Hadronyche versuta. U-ACTX-Hv1a is a positive allosteric modulator of the nicotinic acetylcholine receptor and is also a potent inhibitor of insect voltage-gated Ca2+ receptors. 2+ Channels and voltage-dependent K +The U-ACTX-Hv1a peptide may be a dual antagonist for 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, the disclosures of which are incorporated herein by reference in their entireties. An exemplary U-ACTX-Hv1a peptide is provided in SEQ ID NO:60.

[0232] "U+2 peptide" or "U+2 protein" or "U+2 toxin" or "U+2" or "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 among several peptides, any of which can result in a "U+2 peptide" with unique properties as discussed herein. In some preferred embodiments, the dipeptide is "GS," and an exemplary U+2-ACTX-Hv1a peptide is set forth in SEQ ID NO:61.

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

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

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

[0236] A "vector" refers to a segment of DNA that accepts a foreign gene of interest (e.g., a crip). The gene of interest is also known as the "insert" or "transgene."

[0237] "Vip" or "VIP" or "plant insecticidal protein" refers to a protein discovered by screening supernatants of vegetatively grown strains of Bt for possible insecticidal activity. Vips bear little or no similarity to Cry proteins. Of particular use herein and preferred use is what has been called VIP3 or Vip3 protein, which has lepidopteran activity. Vip is believed to have a similar mode of action as the Bt cry peptide.

[0238] "Vitrification" refers to the process of converting a material into a glassy amorphous material. A glassy amorphous solid may not 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 condition.

[0240] By "yeast expression vector" or "expression vector" or "vector" is meant a plasmid that can introduce a heterologous gene and / or expression cassette that can be transcribed and translated into a yeast cell.

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

[0242] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, a single step, composition of matter, group of steps, or group of compositions of matter shall be considered to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0243] The present disclosure will be practiced without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA techniques, solid phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid phase peptide synthesis, immunology, cell culture, and formulations. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), volumes I, II and III in their entirety; DNA Cloning: A Practical Approach, Vols. I and II (D.N. Glover, ed., 1985), IRL Press, Oxford, all texts; Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed., 1984), IRL Press, Oxford, all texts, and in particular in the articles therein by Gait, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press, Oxford, all texts. 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., 2000) (each of these references is incorporated herein by reference in its entirety).

[0244] Throughout this specification, unless the context requires otherwise, the use of "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated step or element or integer, or group of steps or elements or integers, but not the exclusion of any other step or element or integer, or group of elements or integers.

[0245] All patent applications, patents, and publications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety, and all patent applications, patents, and publications cited herein are incorporated by reference in their entirety, except for any definitions, subject matter disclaimers, or disclaimers, and except to the extent the incorporated material is inconsistent with the disclosure herein, in which case the language of the present disclosure will control.

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

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

[0248] In some embodiments, CRIP can be isolated from Hadronyche versuta or the Blue Mountain funnel-web spider, Hadronyche venenata, Atrax robustus, Atrax formidabilis, or Atrax infensus.

[0249] In some embodiments, the CRIP can 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-nemethoxin-Csp1a, U1-nemethoxin-Csp1b, U1-nemethoxin-Csp1c, U1-plectoxin-Pt1a, U1-plectoxin -Pt1b, U1-plectoxin-Pt1c, U1-plectoxin-Pt1d, U1-plectoxin-Pt1f, U1-serafotoxin-Cv1a, U1-serafotoxin-Hh1a_1, U1-serafotoxin-Hh1a_2, U1-serafotoxin-Hh1a_3, U1-serafotoxin-Hh1b, U1-serafotoxin-Hh1c_1, U1-serafotoxin-Hh1c_2, U1-serafotoxin-Hh1d, U1-serafotoxin-Hh1e, U1-serafotoxin-Hh1f_1, U1-serafotoxin-Hh1f_2, U1-serafotoxin Photoxin-Hh1f_3, U1-Serafotoxin-Hh1f_4, U1-Serafotoxin-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-serafotoxin-Hh1a, U3-sirtautoxin-As1a, U3-plectoxin-Pt1a, U5-ctenitoxin-Pn1a, U7-ctenitoxin-Pk1a, β-hexatoxin-Mg1a, β-hexatoxin-Mr1a, Γ-ctenitoxin-Pn1a, δ-actinopoditoxin-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-serafotoxin-Ec2a, κ-serafotoxin-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 ω-Hexatoxin-Mg2a, μ-Serafotoxin-Hh1a, ω-Actinopositoxin-Mb1a, ω-Agatoxin-Aa4a, ω-Agatoxin-Aa4b, ω-Agatoxin-Aa4c, ω-Hexatoxin-Ar1a1, ω-Hexatoxin-Ar1a3, ω-Hexatoxin-Ar1b1, ω-Hexatoxin-Ar1d1, ω-Hexatoxin-Ar1d4, ω-Hexatoxin-Ar1e1 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-Atracotoxin-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_5ω-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-Hexatoxin-Hv2a, ω-Hexatoxin-Hv2b_1, ω-Hexatoxin-Hv2b_2, ω-Hexatoxin-Hv2b_3, ω-Hexatoxin-Hv2a, 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-Hexatoxin-Hv2a Hexatoxin-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-serafotoxin-Asp1a, ω-serafotoxin-Asp1f, ω-serafotoxin-Asp1g, ω-serafotoxin-Ba1a, ω-serafotoxin-Ba1b, ω-serafotoxin-Bs1a, ω-serafotoxin-Bs2a, or ω-serafotoxin-Hh2a.

[0250] In some embodiments, CRIP can be a spider venom peptide or protein having an amino acid sequence set forth in any one of SEQ ID NOs: 192-278 and 281-370.

[0251] In some embodiments, a polynucleotide encoding CRIP may encode a CRIP having 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 the amino acid sequence set forth in SEQ ID NOs: 192-278 and 281-370.

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

[0253] In some embodiments, the CRIP can 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); U+2-ACTX-Hv1a having the amino acid sequence "SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD" (SEQ ID NO: 62); omega-ACTX-Hv1a having the amino acid sequence "GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD" (SEQ ID NO: 62), "ω+2-ACTX-Hv1a+2" (or "omega+2-ACTX-Hv1a") having the amino acid sequence "GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD" (SEQ ID NO: 63), and kappa+2-ACTX-Hv1a (or κ+2-ACTX-Hv1a) having the amino acid sequence "GSAICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP" (SEQ ID NO: 64).

[0255] In some embodiments, CRIP can 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 comprise 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 to SEQ ID NOs: 60-64, 192-370, and 594.

[0257] In some embodiments, a polynucleotide encoding an ACTX peptide can encode an ACTX peptide having 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 the amino acid sequence set forth in SEQ ID NOs: 60-64 and 594.

[0258] CNTX-Pn1a peptide In some preferred embodiments, the CRIP can be a Γ-CNTX-Pn1a or γ-CNTX-Pn1a toxin. The Γ-CNTX-Pn1a peptide is an insecticidal neurotoxin derived from the Brazilian armoured spider Phoneutria nigriventer. Γ-CNTX-Pn1a targets the N-methyl-D-aspartate (NMDA) subtype ionotropic 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 No. P59367). A recombinant mature Γ-CNTX-Pn1a peptide is provided having the amino acid sequence "GSCADINGACKSDCDCCGDSVTCDCYWSDSCKCRESNFKIGMAIRKKFC" (SEQ ID NO: 65).

[0259] In some embodiments, the Γ-CNTX-Pn1a peptide can comprise an amino acid sequence having 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% amino acid sequence identity to SEQ 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 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 100% identical, at least 101% identical, at least 102% identical, at least 103% identical, at least 104% identical, at least 105% identical, at least 106% identical, at least 107% identical, at least 108% identical, at least 109% identical, at least 110% identical, at least 111% identical, at least 112% identical, at least 113% identical, at least 114% identical, at least 115% identical, at least 116% identical, at least 117% identical, at least 118% identical, at least 119% identical, at least 120% identical, at least 121% identical, at least 122% identical, at least 123% identical, at least 124% identical, at least 125% identical, at least 126% identical, at least 127% identical, at least 128% identical, at least 129% identical, at least 130% identical, at least The Γ-CNTX-Pn1a peptide can be encoded 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 that is a member of the Agelenidae family, i.e., funnel web weavers. 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. Hobo spider venom has been implicated in 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 Agelenidae family, produce venom containing agatoxins, which exhibit insecticidal activity. Agatoxins are a chemically diverse group of toxins that can induce various insecticidal effects depending on the target species. For example, agatoxins cause delayed spastic paralysis in Coleoptera, Lepidoptera, and Diptera, increase the rate of neuronal firing in the central nervous system (CNS) of houseflies (Musca domestica), and are lethal to other insects (e.g., blowflies, Lucilia cuprina). Thus, agatoxins are involved in CNS targeting. 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 include U1-agatoxin-Ta1a and U1-agatoxin-Ta1b, both members of the helical arthropod neuropeptide-derived (HAND) toxin family. In addition to spiders, these toxins are also found in the venom of centipedes. Agatoxins are evolutionary descendants of an ancient ecdysozoan hormone family, the ion transport peptide / crustacean hyperglycemic 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 U1-agatoxin-Ta1b toxin has the full-length amino acid sequence "MKLQLMICLVLLPCFFCEPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQKG (SEQ ID NO: 48)," which includes a signal peptide from amino acid positions 1-17 and the mature toxin from positions 18-68. Id. The protein contains four tightly packed alpha helices with no beta strands, and the molecular weight of the mature toxin is 5700.39 daltons (Da). Id.

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

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

[0267] A U1-agatoxin-Talb variant polypeptide (TVP) is a mutant or variant that differs in some way from wild-type U1-agatoxin-Talb (SEQ ID NO: 1). For example, in some embodiments, this difference can be an amino acid substitution, deletion, or addition, or a change relative to the polynucleotide encoding wild-type U1-agatoxin-Talb that can result in an amino acid substitution, deletion, or addition. The result of this variation is a non-naturally occurring polypeptide and / or its encoding polynucleotide sequence that has enhanced insecticidal activity against one or more insect species compared to wild-type U1-agatoxin-Talb.

[0268] In some embodiments, TVP can have an amino acid sequence according to 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, a polynucleotide sequence having a sequence according to SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654 is operable to encode TVP. For example, in some embodiments, the polynucleotides set forth in Table 2 are operable 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 An exemplary description of TVP and polynucleotides operable to encode TVP is provided in International Application No. PCT / US21 / 28254, the disclosure of which is incorporated herein by reference in its entirety.

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

[0272] 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, 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, at least 99.10% identical, at least 99.11% identical, at least 99.12% identical, at least 99.13% identical, at least 99.14% identical, at least 99.15% identical, at least 99.16% identical, at least 99.17% identical, at least 99.18% identical, at least 99.19 ... 9.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide is the wild-type form of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. 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 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, 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, or at least 99.8% identical to the amino acid sequence according to formula (I): 1. A TVP comprising an amino acid sequence that is at least 99.9% identical, or 100% identical, to: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1, and 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 at X1, X2, X3, X4, or X5).

[0274] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) has an amino acid sequence 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, or at least and X1 is A, S, or a pharmaceutically acceptable salt thereof. 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, TVP has one amino acid substitution at X1, X2, X3, X4, or X5, and X7 is glycine).

[0275] 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, 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 or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide contains at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1, and X1 is A, S, or 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, and TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is absent).

[0276] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) has an amino acid sequence 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 and a TVP comprising the amino acid sequence EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, or a pharmaceutically acceptable salt thereof, which is 99.9% identical or 100% identical to the 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 set forth in SEQ ID NO: 1, and 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, and TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X6 and X7 are absent).

[0277] In some embodiments, the insecticidal U1-agatoxin-Talb 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 to an amino acid sequence according to formula (I): or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Talb set forth in SEQ ID NO: 1, wherein X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, V, M, X3 is I, C, E, T, or S, 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 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 15, 49 to 53, 621 to 622, 624 to 628, 631 to 640, 642 to 651, or 653 to 654).

[0278] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) has an amino acid sequence 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 to an amino acid sequence according to the following formula (I): or a TVP comprising a 100% identical 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 set forth in SEQ ID NO: 1, and X1 is A, S, or N, and X2 is R, Q, N, or a pharmaceutically acceptable salt thereof. , 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 encoded by a polynucleotide sequence set forth in any one of SEQ ID NOs: 17 to 30, 54 to 58, or 655 to 688).

[0279] 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, 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 to an amino acid sequence according to the following formula (I): The TVP may comprise 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 comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1, and wherein X1 is A, S, or N, and X2 is R, Q, or N. , 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, and the TVP further comprises a homopolymer or heteropolymer of two or more TVPs, wherein the amino acid sequence of each TVP is the same or different).

[0280] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) has an amino acid sequence 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 to the amino acid sequence according to formula (I): The TVP may comprise an amino acid sequence that is 100% identical to or 100% identical to: 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-Talb set forth in SEQ ID NO: 1, and wherein X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, 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 the TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each TVP can be the same or different).

[0281] In some embodiments, the insecticidal U1-agatoxin-Talb 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 to an amino acid sequence according to formula (I): and X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S. The TVP may be a TVP comprising the 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 set forth in SEQ ID NO: 1, and wherein 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, and X7 is G or absent; the TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each TVP can be the same or different, and the linker is cleavable in the gut or hemolymph of the insect.

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

[0283] 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, 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 to an amino acid sequence according to formula (I): or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence that is at least 0.8%, at least 99.9% identical, or 100% identical to: EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide has at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1. 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, X7 is G or absent, and Z1 is T or S, then the TVP is glycosylated.

[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, 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 100% identical, at least 101% identical, at least 102% identical, at least 103% identical, at least 104% identical, at least 105% identical, at least 106% identical, at least 107% identical, at least 108% identical, at least 109% identical, at least 110% identical, at least 111% identical, at least 112% identical, at least 113% identical, at least 114% identical, at least 115% identical, at least 116% identical, at least 117% identical, at least 118% identical, at least 119% identical, at least 120% identical, at least 121% identical, at least 122% identical, at least 123% identical, at least 124% identical, at least 125% identical, at least 126% identical, at least 127% identical, at least 128% identical, at least 129% identical, at least 130% The TVP may be a TVP comprising 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, or at least 95% identical to the amino acid sequence according to formula (II): First, it may be a TVP comprising 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 to: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1, and X1 is R or Q, and Z1 is T or A.

[0286] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) has an amino acid sequence 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 100% identical to an amino acid sequence according to the following formula (II): or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence that is 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: 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 set forth in SEQ ID NO: 1, X1 is R or Q, Z1 is T or A, and when Z1 is T, the TVP is glycosylated).

[0287] In some embodiments, the insecticidal U1-agatoxin-Talb 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 to an amino acid sequence according to the following formula (II): % 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: EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, or a pharmaceutically acceptable salt thereof, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b set forth in SEQ ID NO: 1, and wherein 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, 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 100% identical, at least 101% identical, at least 102% identical, at least 103% identical, at least 104% identical, at least 105% identical, at least 106% identical, at least 107% identical, at least 108% identical, at least 109% identical, at least 110% identical, at least 111% identical, at least 112% identical, at least 113% identical, at least 114% identical, at least 115% identical, at least 116% identical, at least 117% identical, at least 118% identical, at least 119% identical, at least 120% identical, at least 121% identical, at least 122% identical, at least 123% identical, at least 124% identical, at least 125% identical, at least 126% identical, at least 127% identical, at least 128% identical, at least 129% identical, at least 130% or a pharmaceutically acceptable salt thereof.

[0289] In some embodiments, 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 100% identical, at least 101% identical, at least 102% identical, at least 103% identical, at least 104% identical, at least 105% identical, at least 106% identical, at least 107% identical, at least 108% identical, at least 109% identical, at least 110% identical, at least 111% identical, at least 112% identical, at least 113% identical, at least 114% identical, at least 115% identical, at least 116% identical, at least 117% identical, at least 118% identical, at least 119% identical, at least 120% identical, at least 121% identical, at least 122% identical, at least 123% identical, at least 124% identical, at least 125% identical, at least 126% identical, at least 127% identical, at least 128% identical, at least 129% identical, at least 130% identical, at least 131% identical, at least 132% identical % 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: "EPDEICRAQMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYAACHEAQKG" (SEQ ID NO: 51).

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

[0291] In various embodiments, a polynucleotide encoding TVP can be used to transform plant, yeast, or bacterial cells. In some embodiments, the insecticidal TVP transgenic protein can be formulated into a composition that can be sprayed or otherwise applied to the surface of a plant or part thereof in any manner known to those of skill in the art. Accordingly, provided herein are DNA constructs operable to encode one or more TVPs under appropriate conditions in a host cell, e.g., a plant cell. A method for controlling pest infection of a plant cell by a parasitic insect includes recombinantly administering or introducing a TVP-encoding polynucleotide described herein into a plant, plant tissue, or plant cell, and growing the recombinantly modified plant, plant tissue, or plant cell in a field where it is exposed to the pest. Alternatively, TVP can be formulated into a sprayable composition consisting of TVP and an excipient, which can be applied directly to a susceptible plant by direct application to produce a deleterious effect upon ingestion of the TVP by an infective insect.

[0292] Scorpion peptides and toxins In some embodiments, CRIP can 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 (limbatoxin), potassium channel toxin α-KTx1.7 (Lqh15-1), potassium channel toxin α-KTx1.9 (hongotoxin-2), potassium channel toxin α-KTx1.10 (parabutoxin-3), potassium channel toxin α-KTx1.11 (throtoxin), potassium channel toxin α-KTx1.13 (charybdotoxin 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 (noxiustoxin-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 (karyotoxin-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 (charybdotoxin-like peptide Bs6), potassium channel toxin α-KTx3.9 (karyotoxin-3), potassium channel toxin α-KTx4.1 (titutoxin K-α), potassium channel toxin α-KTx4.3 (toxin TdK1), potassium channel toxin α-KTx4.4 (toxin Tc30), potassium channel toxin α-KTx5.1 (leurotoxin-1), potassium channel toxin α-KTx5.2 (leurotoxin-like toxin P05), potassium channel toxin α-KTx5.4 (tamapin), potassium channel toxin α-KTx5.5 (tamapin) Mapin-2), potassium channel toxin α-KTx6.1 (potassium channel blocking toxin 1), potassium channel toxin α-KTx6.2 (maurotoxin), potassium channel toxin α-KTx6.3 (neurotoxin HsTX1), potassium channel toxin α-KTx6.12 (anuroctoxin), potassium channel toxin α-KTx6.13 (spinoxin), potassium channel toxin α-KTx6.14 (HgeTx1), potassium channel toxin α-KTx7.2 ( 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), insectoxin-I1, small toxin (peptide I), insectoxin-I3 (BeI3), insectoxin-I4 (BeI4), insectoxin-I5A, neurotoxin 8 (neurotoxin VIII), putative toxin Lqh8 / 6, neurotoxin 9 (neurotoxin IX), maurocalcine (MCa), chlorotoxin-like peptide Bs14 (Bs14), chlorotoxin (CTX), neurotoxin P2, insectoxin-I5 (BeI5), potassium channel toxin α-KTx6.15 (hemitoxin), toxin GaTx1, AahIT1, phaodotoxin, 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 can be a scorpion peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 88-191.

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

[0295] In some embodiments, the CRIP can 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 can be an AaIT1 toxin. The protein toxin AalT1 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 according to SEQ ID NO: 88 (NCBI accession number P01497.2). AaIT1 is a site 4 toxin that forces insect sodium channels to open by lowering the activation reaction energy barrier.

[0297] In some embodiments, the scorpion peptide may comprise 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 to SEQ ID NOs: 66, 88-191.

[0298] In some embodiments, a polynucleotide encoding a scorpion peptide or toxin can encode a scorpion peptide or toxin having 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 the amino acid sequence set forth in SEQ ID NOs: 66, 88-191.

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

[0300] In some embodiments, CRIP can be one of the following sea anemone toxins: toxin AETX-1 (AETX I), toxin APETx1, toxin APETx2, antihypertensive protein BDS-1 (antihypertensive I), antihypertensive protein BDS-2 (antihypertensive 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), anthropoiulin-A (toxin AP-A), anthropoiulin-B ( toxin AP-B), anthopleurin-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 can be a sea anemone peptide having the amino acid sequence set forth 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, which has a variety of toxins that it uses to defend itself. One of the toxins derived from Anemonia viridis is the neurotoxin "Av3." Av3 inhibits voltage-dependent sodium (Na) receptor site 3. + Av3 is a type III sea anemone toxin that inhibits the inactivation of the sodium channel, resulting in contractile paralysis. Binding of the Av3 toxin to site 3 inactivates and destabilizes the sodium channel, causing it to remain 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 exhibits high selectivity for crustacean and insect sodium channels and low selectivity for mammalian sodium channels (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 is provided, having the amino acid sequence of SEQ ID NO:44.

[0303] In some embodiments, a CRIP of the present invention can be an Av3 variant polypeptide (AVP). In some embodiments, the AVP may have the following amino acid variations from SEQ ID NO: 44: an N-terminal amino acid substitution of R1K compared to SEQ ID NO: 44 (changing the polypeptide sequence from the wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "KSCCPCYWGGCPWGQNCYPEGCSGPKV" (SEQ ID NO: 45)), a C-terminal amino acid deletion compared to SEQ ID NO: 44 (changing the polypeptide sequence from the wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "RSCCPCYWGGCPWGQNCYPEGCSGPK" (SEQ ID NO: 46)), and / or an N-terminal mutation and a C-terminal mutation (changing the polypeptide sequence from the wild-type "RSCCPCYWGGCPWGQNCYPEGCSGPKV" to "KSCCPCYWGGCPWGQNCYPEGCSGPK" (SEQ ID NO: 47) where the N-terminal amino acid may have a substitution of R1K 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 variants thereof are described in applicant's PCT application (International Application No. PCT / US19 / 51093), filed September 13, 2019, entitled "Av3 Variant Insecticidal Polypeptides and Methods of Making and Using Same," the disclosure of which and the disclosure of Av3 peptides or variants thereof are set forth and are incorporated herein by reference in their entirety.

[0305] In some embodiments, the sea anemone peptide can comprise 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 to SEQ ID NOs:44-47 and 371-411.

[0306] In some embodiments, the polynucleotide encoding the sea 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, or at least 88% identical to the amino acid sequence set forth in SEQ ID NOs: 44-47 and 371-411. , 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 Peptides and Conotoxins Conotoxins are toxins isolated from cone snails that act by disrupting neuronal communication. Examples of conotoxins include α-, ω-, μ-, δ-, and κ-conotoxins. Briefly, α-conotoxins (and αA- and φ-conotoxins) target nicotinic ligand-gated channels, ω-conotoxins target voltage-gated calcium channels, μ-conotoxins target voltage-gated sodium channels, δ-conotoxins target voltage-gated sodium channels, and κ-conotoxins target voltage-gated potassium channels.

[0308] In some embodiments, CRIP can be isolated from an organism belonging to the genus Conus, and the isolated peptide is a conotoxin.

[0309] In some embodiments, CRIP may be 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, a CRIP can be a toxin, peptide, or protein (also known as a venom or venom peptide or protein) produced and / or isolated from an arthropod, spider, scorpion, insect, bee, wasp, centipede, crustacean, reptile, snake, lizard, amphibian, frog, salamander, mollusk, cone snail, cnidarian, sea anemone, jellyfish, hydrozoan, cephalopod, octopus, squid, cuttlefish, fish, or mammal.

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

[0312] CRIP-insecticidal protein A CRIP-insecticide protein is any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement consisting of: (1) at least one CRIP or two or more CRIPs, and (2) additional non-CRIP peptides, polypeptides, or proteins (e.g., in some embodiments, having the ability to: increase mortality and / or inhibit insect growth when insects are exposed to the CRIP-insecticide protein compared to CRIP alone, increase expression of the CRIP-insecticide protein, e.g., in a host cell or expression system, and / or affect post-translational processing of the CRIP-insecticide protein).

[0313] In some embodiments, a CRIP-insecticidal protein can be a polymer comprising 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, a CRIP-insecticidal protein can refer to one or more CRIPs operably linked to one or more proteins such as a stabilization domain (STA), an endoplasmic reticulum signaling protein (ERSP), an insect-cleavable or insect-non-cleavable linker (L), and / or any other combination thereof.

[0315] In some embodiments, the CRIP-insecticidal protein may be a non-naturally occurring protein comprising (1) wild-type CRIP and (2) an additional peptide, polypeptide, or protein (e.g., ERSP, a linker, STA, UBI, or a histidine tag or similar marker).

[0316] In some embodiments, the CRIP-insecticidal protein can be a non-naturally occurring protein, including (1) wild-type CRIP and (2) a non-naturally occurring CRIP.

[0317] In some embodiments, the CRIP-insecticidal protein may be a non-naturally occurring protein comprising (1) wild-type CRIP, (2) non-naturally occurring CRIP, and (3) an additional peptide, polypeptide, or protein (e.g., ERSP, a linker, STA, UBI, or a histidine tag or similar marker).

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

[0319] In some embodiments, an insecticidal protein can comprise one or more CRIPs disclosed herein. In some embodiments, an insecticidal protein can comprise a CRIP homopolymer (e.g., two or more CRIP monomers where the CRIP is the same). In some embodiments, an insecticidal protein can comprise a CRIP heteropolymer (e.g., two or more CRIP monomers where the CRIP monomers are different).

[0320] In some embodiments, the insecticidal protein can comprise a fusion protein comprising two or more CRIPs separated by cleavable or non-cleavable linkers, and the amino acid sequence of each CRIP can be the same or different.

[0321] In some embodiments, the insecticidal protein may comprise a fusion protein comprising two or more CRIPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRIP may be the same or different, and the linker is cleavable within the insect gut or hemolymph.

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

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

[0324] Methods for Producing CRIP or Peptide-IA Methods for producing proteins are well known in the art, and a variety of techniques are available. For example, in some embodiments, proteins can be produced using recombinant methods or chemically synthesized. The present disclosure provides methods for producing CRIP, CRIP-insecticide proteins, and other peptide insecticides (peptide-IAs). These methods are described in detail below.

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

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

[0327] In some embodiments, the invention comprises, consists essentially of, or consists of a method for producing CRIP, the method comprising: (a) preparing a vector comprising a first expression cassette comprising, consisting essentially of, or consisting of a polynucleotide operable to encode CRIP, or a complementary nucleotide sequence thereof; (b) introducing the vector into a host cell (e.g., a bacterium or yeast, or an insect, or plant cell, or an animal cell); and (c) growing the yeast strain in a culture medium under conditions operable to allow 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 practiced in a wide variety of host cells (see the Host Cells section below). Indeed, end users of the present invention can practice the teachings in any host cell of their choice. Thus, in some embodiments, the host cell can be any host cell that meets the requirements of the end user; i.e., in some embodiments, expression of CRIP can be achieved using a variety of host cells and based on the teachings herein. For example, in some embodiments, a user may desire to use one particular type of host cell (e.g., yeast cells or bacterial cells) as opposed to another, and preferences for a given host cell can range from availability to cost.

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

[0330] Isolation and mutagenesis of wild-type CRIP A CRIP or peptide-insecticide (peptide-IA) can be obtained directly from a source (e.g., by isolating the CRIP or peptide-IA from an animal). A mutant CRIP or peptide-IA can be produced by making a mutation in a wild-type CRIP or peptide-IA polynucleotide sequence, inserting the CRIP or peptide-IA polynucleotide sequence into an appropriate vector, transforming a host organism in a manner such that the polynucleotide encoding CRIP or peptide-IA is expressed, culturing the host organism to produce a desired amount of CRIP or peptide-IA, and then purifying the CRIP or peptide-IA from in and / or around the host organism.

[0331] Generation of mutations in wild-type CRIP or Peptide-IA polynucleotide sequences can be achieved by a variety of means well known to those skilled in the art, including the Kunkel method, cassette mutagenesis, PCR site-directed mutagenesis, the "perfect murder" technique (delitto perfetto), 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 regions of homology, the "pop-in pop-out" method, and CRISPR-Cas 9.Exemplary methods for site-directed mutagenesis are described in 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 U S A. 1982 Nov;79(21):6409-13; Bachman, Site-directed mutagenesis. Methods Enzymol. 2013;529:241-8; Carey et al., PCR-mediated 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 (the disclosures of all of the foregoing references are incorporated herein by reference in their entireties).

[0332] Wild-type CRIP, e.g., spider toxins, scorpion toxins, and / or other toxins, can be isolated from venom. For example, spider venom can be isolated from the venom gland of a spider (e.g., a spider such as Eratigena agrestis) using any of the techniques known to those skilled in the art. For example, in some embodiments, venom can be isolated from a spider according to the methods described in U.S. Patent No. 5,688,764, the disclosure of which is incorporated herein by reference in its entirety.

[0333] Wild-type CRIP or Peptide-IA polynucleotide sequences can be obtained by screening genomic libraries using primer probes directed to the CRIP or Peptide-IA polynucleotide sequence. 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 generated using oligonucleotide synthesis methods such as the phosphoramidite, triester, phosphite, or H-phosphonate methods. See Engels, J. W. and Uhlmann, E. (1989), Gene Synthesis (New Synthetic Methods (77)). Angew. Chem. Int. Ed. Engl., 28:716-734, the disclosure of which 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 can be chemically synthesized using commercially available polynucleotide synthesis services, such as those offered by GENEWIZ® (e.g., TurboGENE™; PriorityGENE; and FragmentGENE) or SIGMA-ALDRICH® (e.g., custom DNA and RNA oligo designs and custom DNA oligo orders). Exemplary methods for generating DNA and / or custom chemically synthesized polynucleotides are well known in the art and are illustratively provided in U.S. Pat. No. 5,736,135, filed February 13, 1995 (Serial No. 08 / 389,615), the disclosure of which 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 Publishers, Amsterdam, NL, vol. 48, No. 12, 1992, pp. 2223-2311; see also Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties; Methods in Molecular Biology Vol. 20, the disclosure of which is incorporated herein by reference in its entirety.

[0335] Chemically synthesized polynucleotides allow for the production of DNA sequences tailored to produce a desired polypeptide 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 an amino acid sequence in which each amino acid corresponds to a codon in the mRNA sequence. Thus, the amino acid composition of a polypeptide chain translated from an mRNA sequence can be changed by altering the underlying codons that determine which of the 20 amino acids are added to the growing polypeptide. Thus, mutations in DNA, such as insertions, substitutions, deletions, and frameshifts, can result in amino acid insertions, substitutions, or deletions, depending on the underlying codons.

[0336] Obtaining CRIP or Peptide-IA from a chemically synthesized DNA polynucleotide sequence and / or a wild-type DNA polynucleotide sequence modified through mutagenesis can be accomplished by cloning the DNA sequence into an appropriate vector. A variety of expression vectors, host organisms, and cloning strategies are available, and are known to those skilled in the art. For example, a vector can be a plasmid, allowing the introduction of a heterologous gene and / or expression cassette to be transcribed and translated 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 where it can be replicated. A vector can contain "vector elements," such as an origin of replication (ORI), a gene conferring antibiotic resistance to allow selection, multiple cloning sites, a promoter region, a selectable marker for non-bacterial transfection, and a primer binding site. A nucleic acid sequence can 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 in a location within the host cell's nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One skilled in the art would be well equipped to construct vectors through standard recombinant techniques as described in Sambrook et al., 1989 and Ausubel et al., 1996, both of which are incorporated herein by reference. In addition to encoding a CRIP or Peptide-IA polynucleotide, the vector can encode a targeting molecule. A targeting molecule directs the desired nucleic acid to a particular tissue, cell, or other location.

[0337] Vectors and transformation In some embodiments, a CRIP or Peptide-IA polynucleotide can be cloned into a vector using a variety of cloning strategies and commercially available cloning kits and materials readily available to those skilled in the art. For example, a CRIP or Peptide-IA polynucleotide can be cloned into a vector using a strategy (e.g., SnapFast, Gateway, TOPO, Gibson, LIC, InFusionHD, or Electra strategy). Many vectors are commercially available and can be used to produce CRIP or Peptide-IA. For example, CRIP or Peptide-IA polynucleotides can be generated using polymerase chain reaction (PCR) in combination with the pCR™ II-TOPO vector or PCR™ 2.1-TOPO® vector (commercially available as the TOPO® TA Cloning® Kit from Invitrogen) for 5 minutes at room temperature; the TOPO® reaction can then be transformed into competent cells, followed by selection based on a color change (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; see also Adams et al. Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997, the disclosure of which is incorporated herein by reference in its entirety).

[0338] In some embodiments, a polynucleotide encoding CRIP or Peptide-IA may be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal virus, and plant virus), and / or artificial chromosome (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 a host by digesting approximately 2-5 μg of vector DNA with the necessary restriction enzymes to allow for 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 self-ligation / recircularization). The digested vector is then gel-purified. The desired DNA segment (e.g., a polynucleotide encoding CRIP or Peptide-IA) is then amplified via PCR, and any excess enzymes, primers, unincorporated dNTPs, truncated PCR products, and / or salts are removed from the PCR reaction using techniques known to those skilled in the art (e.g., by using a PCR cleanup kit). The DNA segment of interest is ligated to the vector by preparing a mixture containing approximately 20 ng of vector, approximately 100-1,000 ng or the DNA segment of interest, 2 μL of 10x buffer (i.e., 30 mM Tris-HCl, 4 mM MgCl2, 26 μM NAD, 1 mM DTT, 50 μg / ml BSA, pH 8; store at 25°C), and 1 μL of T4 DNA ligase, and adding HO 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 transformed into competent cells, for example, by electroporation or chemical methods. Colony PCR can then be performed to identify vectors containing the DNA segment of interest.

[0340] In some embodiments, a polynucleotide encoding CRIP or Peptide-IA, together 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 can begin with a signal peptide sequence, followed by a DNA sequence encoding a Kex2 cleavage site (lysine-arginine), followed by a CRIP or Peptide-IA polynucleotide transgene (with a glycine-serine codon at the 5' end and, finally, a stop codon at the 3' end). All of these elements are then expressed as a single open reading frame (ORF) into a fusion peptide in yeast cells. The alpha-mating factor (αMF) signal sequence is most frequently used to facilitate metabolic processing of recombinant insecticidal peptides through the endogenous secretory pathway of recombinant yeast; i.e., the expressed fusion peptide typically enters the endoplasmic reticulum, where the α-mating factor signal 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, and the mature polypeptide (i.e., CRIP or peptide-IA) is then 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 frequency of naturally occurring codons observed in the endogenous open reading frames of a given host organism does 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, codon optimization should be considered for the CRIP or peptide-IA expression ORF, which contains sequence elements encoding a signal sequence, a Kex2 cleavage site, and CRIP or peptide-IA, since they are initially translated as a single fusion peptide in recombinant yeast cells.

[0342] In some embodiments, the 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 and integrating vectors, which are typically designed for specific yeast strains. Careful selection of an appropriate expression vector is required, taking into account the specific yeast expression system used for peptide production. In some embodiments, an integrating vector can be used, which integrates into the chromosome of the transformed yeast cell and is stably maintained throughout cycles of cell division and growth. The integrating DNA sequence is homologous to the target genomic DNA locus in the transformed yeast species; examples of such integration sequences include pLAC4, 25S rDNA, pAOX1, and TRP2. The location of the insecticidal peptide transgene can be adjacent to or within the integrating DNA sequence (insertion vector) or within the integrating DNA sequence (replacement vector).

[0343] In some embodiments, the expression vector may include E. coli elements for DNA preparation in E. coli (e.g., an E. coli origin of replication, an antibiotic selection marker, etc.). In some embodiments, the vector may include a set of sequence elements required for expression of a transgene of interest (e.g., a transcription promoter, a terminator, a yeast selection marker, an integrating DNA sequence homologous to the host yeast DNA, etc.). There are many suitable yeast promoters, including native and engineered promoters (e.g., yeast promoters such as pLAC4, pAOX1, pUPP, pADH1, pTEF, pGal1, and others), that may be used in some embodiments.

[0344] In some embodiments, selection methods such as acetamide prototrophy selection, zeocin resistance selection, geneticin resistance selection, nourseothricin 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 Nos. 6,548,285 (filed April 3, 1997), 6,165,715 (filed June 22, 1998), and 6,110,707 (filed January 17, 1997), the disclosures of which are incorporated herein by reference in their entireties.

[0345] In some embodiments, a polynucleotide encoding CRIP or peptide-IA can be inserted into the pKLAC1 plasmid. pKLAC1 is commercially available from New England Biolabs® Inc. (Item No. (NEB#E1000). pKLAC1 is designed to achieve high-level expression of recombinant proteins (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 contains an MCS downstream of the αMF secretion signal. The αMF secretion signal directs the recombinant protein into the secretory pathway, where it is then cleaved by Kex2 to yield, 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 No. 450-45).

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

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

[0348] In some embodiments, a polynucleotide encoding TVP can be inserted into other commercially available plasmids and / or vectors readily available to one of skill 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 express CRIP at a concentration 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, or at least 190 mg / L per liter of medium in yeast culture. 200mg / L, at least 500mg / L, at least 750mg / L, at least 1,000mg / L, at least 1,250mg / L, at least 1,500mg / L, at least 1,750mg / L, at least 2,000mg / L, at least 2,500mg / L, at least 3,000mg / L, at least 3,500mg / L, at least 4,000mg / L, at least 4,500mg / L, at least 5,000mg / L, at least 5,500mg / L, at least 6,000mg / L, at least 6,500mg / L, at least 7,000mg / L, at least 7,500mg / L, at least 8,000mg / L, at least 8,500mg / L, at least 9,000mg / L, at least 9,500mg / L, at least 10,000mg / L, at least In some embodiments, a CRIP yield 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 can be produced.

[0350] In some embodiments, one or more expression cassettes comprising a polynucleotide operable to express CRIP are inserted into a vector to express CRIP at about 100 mg / L to about 100,000 mg / L, about 110 mg / L to about 100,000 mg / L, about 120 mg / L to about 100,000 mg / L, about 130 mg / L to about 100,000 mg / L, about 140 mg / L to about 100,000 mg / L, about 150 mg / L to about 100,000 mg / L, about 160 mg / L to about 100,000 mg / L, about 170 mg / L to about 100,000 mg / L, or about 180 mg / L to about 100,000 mg / L. 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~Approx. 100,000mg / L, Approx. 8500mg / L~Approx. 100,000mg / L, Approx. 9000mg / L~Approx. 100,000mg / L, Approx. 9500mg / L~Approx. 100,000mg / L, Approx. 10000mg / L~Approx. 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, 000 mg / L, or a range of about 99,500 mg / L to about 100,000 mg / L of CRIP.

[0351] In some embodiments, one or more expression cassettes comprising a polynucleotide operable to express CRIP are inserted into a vector to express CRIP at about 100 mg / L per liter of medium (yeast fermentation supernatant) to about 100,000 mg / L, about 100 mg / L to about 99,500 mg / L, about 100 mg / L to about 99,000 mg / L, about 100 mg / L to about 98,500 mg / L, about 100 mg / L to about 98,000 mg / L, about 100 mg / L to about 97,500 mg / L, about 100 mg / L to about 97,000 mg / L, or about 100 mg / L to about 96,500 mg / L. 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.The yield of CRIP can be in the range of about 100 mg / L to about 2500 mg / L, about 100 mg / L to about 2000 mg / L, about 100 mg / L to about 1500 mg / L, about 100 mg / L to about 1000 mg / L, about 100 mg / L to about 1000 mg / L, about 100 mg / L to about 750 mg / L, about 100 mg / L to about 500 mg / L, about 100 mg / L to about 250 mg / L, about 100 mg / L to about 100 mg / L, or about 100 mg / L to about 110 mg / L.

[0352] In addition to the DNA polynucleotide sequence encoding CRIP or Peptide-IA, additional known DNA segments, known as regulatory elements, can be cloned into the vector to allow for enhanced expression of foreign DNA or transgenes. Examples of such additional DNA segments include: (1) promoter, terminator, and / or enhancer elements, (2) appropriate mRNA stabilization polyadenylation signals, (3) internal ribosome entry sites (IRES), (4) introns, and (5) post-transcriptional regulatory elements. The combination of a DNA segment of interest with any one of the aforementioned cis-acting elements is referred to as an "expression cassette."

[0353] A single expression cassette can include one or more of the aforementioned regulatory elements and a polynucleotide operable to express CRIP or Peptide-IA. For example, in some embodiments, a CRIP or Peptide-IA expression cassette can include a polynucleotide operable to express CRIP or Peptide-IA, an α-MF signal, a Kex2 site, an LAC4 terminator, an ADN1 promoter, and an acetamidase (amdS) selectable marker adjacent to the LAC4 promoter on 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 comprising a polynucleotide operable to express CRIP or Peptide-IA. In alternative embodiments, there are two expression cassettes operable to encode CRIP or Peptide-IA (i.e., a dual expression cassette). In other embodiments, there are three expression cassettes operable to encode 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 express CRIP at a concentration 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, at least 190 mg / L 200mg / L, at least 500mg / L, at least 750mg / L, at least 1,000mg / L, at least 1,250mg / L, at least 1,500mg / L, at least 1,750mg / L, at least 2,000mg / L, at least 2,500mg / L, at least 3,000mg / L, at least 3,500mg / L, at least 4,000mg / L, at least 4,500mg / L, at least 5,000mg / L, at least 5,500mg / L, at least 6,000mg / L, at least 6,500mg / L, at least 7,000mg / L, at least 7,500mg / L, at least 8,000mg / L, at least 8,500mg / L, at least 9,000mg / L, at least 9,500mg / L, at least 10,000mg / L, at least 11,000mg CRIP or peptide-IA can be produced with a yield 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 operable to express CRIP or peptide-IA can be inserted into a vector (e.g., a pKlac1 plasmid) to result in a yield of about 100 mg / L of CRIP or peptide-IA (yeast fermentation broth supernatant). For example, in some embodiments, two expression cassettes containing polynucleotides operable to express CRIP or peptide-IA can be inserted into a vector (e.g., a pKS482 plasmid) to result in a yield of about 2 g / L of CRIP or peptide-IA (yeast fermentation broth supernatant). Alternatively, in some embodiments, three expression cassettes containing polynucleotides operable to express CRIP or peptide-IA can be inserted into a vector (e.g., a pKlac1T plasmid).

[0359] In some embodiments, multiple CRIP or Peptide-IA expression cassettes can be transfected into yeast to allow for integration of one or more copies of the 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: The DNA sequence of a CRIP or Peptide-IA expression cassette is synthesized, including an intact LAC4 promoter element, a codon-optimized CRIP or Peptide-IA expression ORF element, and a pLAC4 terminator element. The intact expression cassette is ligated between the Sal I and Kpn I restriction sites of the pKlac1 vector, downstream of the pLAC4 terminator of pKS477, to obtain the double-transgene CRIP or Peptide-IA expression vector pKS482. The double-transgene vector pKS482 is then linearized using Sac II restriction endonuclease and transformed into the K. lactis YCT306 strain by electroporation. The resulting yeast colonies are then grown on YCB agar plates supplemented with 5 mM acetamide (only acetamidase-expressing cells can efficiently use acetamide as a metabolic source of nitrogen). Approximately 100–400 colonies can be picked from the pKS482 yeast plates to evaluate the yeast colonies. An inoculum from each colony is cultured in 2.2 mL of K. lactis defined medium supplemented with 2% sugar alcohol as a carbon source. The cultures are incubated at 23.5°C with shaking at 280 rpm for 6 days, at which point the cell density in the culture reaches a maximum level, as indicated by optical density 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 (conditioned medium) is filtered through a 0.2 μM membrane for HPLC yield analysis.

[0360] Chemically synthesizing peptides Peptide synthesis or chemical synthesis or peptides and / or polypeptides can be used to generate CRIP or Peptide-IA. These methods can be performed 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 accomplished by using a strategy in which the carboxyl group of a subsequent amino acid is coupled to the N-terminus of a preceding amino acid to generate a nascent polypeptide chain (a process opposite to the type of polypeptide synthesis found in nature).

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

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

[0364] The required C-terminal protecting agent 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 to act as a protecting group. Side chain amino acids require the use of several different protecting groups, which vary based 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 displaced by nucleophilic attack via the primary amino group on the N-terminus of the growing peptide chain. The reactive intermediate generated by the carbodiimide can result in racemization of the amino acid. 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 can be used include 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with an additional activated base, and benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). Finally, deprotection and coupling of the amino acid follow.

[0366] At the end of the synthesis process, removal of protecting groups from the polypeptide (a process that typically occurs via acidolysis) must be performed. The determination of which reagents are required for peptide cleavage depends on the protection scheme used and the overall synthetic methodology. For example, in some embodiments, hydrogen bromide (HBr), hydrogen fluoride (HF), or trifluoromethanesulfonic acid (TFMSA) can be used to cleave Bzl and Boc groups. Alternatively, in other embodiments, less strong acids such as TFA can effect acidolysis of tBut and Fmoc groups. Finally, peptides can be purified based on their physicochemical 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.278, U.S. Patent Nos. 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), and U.S. Patent Application No. 2005 / 0165215 (filed December 23, 2004), the disclosures of which are incorporated herein by reference in their entireties.

[0368] Further exemplary methods for producing polynucleotides, peptides, and CRIPs can be found in U.S. Patent Application Publication No. 2015 / 0148288A1, the disclosure of which 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-insecticidal proteins, or peptide-IA described herein.

[0370] Cell culture and transformation technologies 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" to describe the process of introducing exogenous and / or heterologous DNA or RNA into bacterial cells, and the term "transfection" for the process of describing the introduction of exogenous and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms "transformation" and "transfection" are used interchangeably, regardless of whether the process describes the introduction of exogenous and / or heterologous DNA or RNA into prokaryotic organisms (e.g., bacteria) or eukaryotic organisms (e.g., yeast, plants, or animals).

[0371] In some embodiments, host cells can be transformed using the following methods: electroporation, cell squeezing, microinjection, impalement, use of hydrostatic pressure, sonoporation, optical transfection, continuous injection, lipofection, by use of viruses (e.g., adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus), chemical phosphate method, DEAE-dextran or polyethyleneimine (PEI)-mediated endocytosis, protoplast fusion, hydrodynamic delivery, magnetofection, nucleoinfection, and / or other methods. Exemplary methods for 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; 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 its entirety).

[0372] Electroporation is a technique in which electricity is applied to cells, permeabilizing the cell membrane and thereby allowing 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 electroporation are commercially available (e.g., Gene Pulser Xcell™ Electroporation System, Bio-Rad®, Neon® Transfection System for Electroporation, Thermo-Fisher Scientific, and other tools and / or devices). Exemplary methods of electroporation are set forth in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3; Saito (2015) Electroporation Methods in Neuroscience. Springer Press; Pakhomov et al., (2017) Advanced Electroporation Techniques in Biology and Medicine. Taylor & Francis (the disclosures of which are incorporated herein by reference in their entireties).

[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 is 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 approximately 10-200 mL of yeast extract peptone dextrose (YEPD) and incubating the yeast culture at 30°C on a shaker until the yeast reaches early logarithmic growth phase (e.g., approximately 0.6-2 x 10 8This can be achieved by incubating the yeast at 3000 rpm for 5 minutes (cells / mL). The yeast are collected in a sterile centrifuge tube and centrifuged at 3000 rpm for 5 minutes at 4°C (Note: Keep cells cold during the procedure), the cells are washed with 40 mL of ice-cold sterile deionized water, and the cells are pelleted at 23,000 rpm for 5 minutes. The wash step is repeated, and the cells are resuspended in 20 mL of 1 M fermentable sugar (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohol (e.g., erythritol, hydrogenated starch hydrolysate, isomalt, lactitol, maltitol, mannitol, and xylitol), followed by spinning down at 3,000 rpm for 5 minutes. The cells are resuspended in an appropriate amount of ice-cold 1 M fermentable sugar (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohol (e.g., erythritol, hydrogenated starch hydrolysate, isomalt, lactitol, maltitol, mannitol, and xylitol) to a final cell density of 3 x 10 9 40 μl of yeast suspension was mixed with approximately 1-4 μl of vector containing a linear polynucleotide (approximately 1 μg) encoding CRIP or peptide-IA in a pre-chilled 0.2 cm electroporation cuvette (Note: Ensure the sample contacts both sides of the aluminum cuvette). A single pulse of 2000 V was applied with the optimal time constant (5 ms) of the RC circuit. The cells were then recovered 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, hydrogenated starch hydrolysate, isomalt, lactitol, maltitol, mannitol, and xylitol) and then spread onto selective plates.

[0374] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding CRIP or peptide-IA into plant protoplasts by incubating sterilized plant material in plant protoplast solution (e.g., approximately 8 mL of 10 mM 2-[N-morpholino]ethanesulfonic acid (MES) (pH 5.5), 0.01% (w / v) pectylase, 1% (w / v) macerozyme, 40 mM CaCl2, and 0.4 M mannitol) and placing the mixture on a rotary shaker for approximately 3-6 hours at 30°C to generate protoplasts. Remove debris by filtration through an 80 μm mesh nylon screen. Rinse the screen with approximately 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 and then centrifuge at approximately 300 x g for approximately 5 minutes. After centrifugation, discard the supernatant and wash with 5 mL of plant electroporation buffer. Place the protoplasts in plant electroporation buffer at approximately 1.5 x 10 per mL of liquid. 6 ~2×10 6 Resuspend the protoplasts in 1 mL of PBS. Transfer approximately 0.5 mL of the protoplast suspension to one or more electroporation cubes, set them on ice, and add the vector. (Note: For stable transformation, the vector is linearized using one of the restriction methods described above, and approximately 1-10 μg of vector is used. For transient expression, the vector may be maintained in its supercoiled state, and approximately 10-40 μg of vector may be used.) Mix the vector and protoplast suspension. Place the cuvette in the electroporation apparatus and shock one or more times at approximately 1-2 kV (initial, a capacitance of 3-25 μF may be used while optimizing the reaction). Return the cuvette to ice and dilute the transformed cells 20-fold in complete medium. After approximately 48 hours, harvest the protoplasts.

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

[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 can be an archaebacterium or a eubacterium, such as a gram-negative or gram-positive organism. Examples of useful bacteria include Escherichia coli (e.g., E. coli), Bacillus (e.g., B. subtilis), Enterobacteriaceae, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.

[0377] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA can be a unicellular cell. For example, in some embodiments, the host cell can 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 cell 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-insecticidal protein, or peptide-IA may be eukaryotic.

[0381] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA can be a cell belonging to the following clades: Opisthokonta, Chlorophyta (e.g., algae and plants), Amoebozoa, Cercozoa, Alveolata, Flagellates, Heterokontha, Dicyclistata, or Excavata.

[0382] In some embodiments, the procedures and methods described herein can be accomplished using a host cell that is, for example, a metazoan, a choanoflagellate, or a fungus.

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

[0384] In some embodiments, the procedures and methods described herein can be accomplished 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 accomplished 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 accomplished using a host cell that is Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, or Pichia pastoris.

[0387] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be a fungus 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 can be a member of the Saccharomycetaceae family. For example, in some embodiments, the host cell can 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 can 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 cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be a 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 can be a species within the genus Kluyveromyces, for example, the host cell can be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.

[0392] In some embodiments, the host cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA can be a species within the genus Pichia. For example, the host cell can 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 cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be a 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 can 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 producing recombinant CRIP or Peptide-IA is an exceptional method known to those skilled in the art. In some embodiments, the methods and compositions described herein can be practiced with any species of yeast (including, but not limited to, any species of the genus Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces), including any species of Saccharomyces, such as Saccharomyces cerevisiae 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 Pichia species include any species in the genus Pichia (e.g., the Pichia species Pichia pastoris), for example, Pichia pastoris 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, and a Pichia selected from the following strains: pastoris: Bg08, X-33, SMD1168, and KM71.In some embodiments, any Kluyveromyces species can be used to accomplish the methods described herein, including any species of the genus Kluyveromyces (e.g., Kluyveromyces lactis), and as taught by the inventors, the strain of Kluyveromyces lactis can be, but is not necessarily selected from, the following strains: 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, in addition to Kluyveromyces lactis species 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 cell 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 cell used to produce CRIP, CRIP-insecticidal protein, or peptide-IA may be Bacillus licheniformis.

[0400] In some embodiments, the host cell 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 accomplished using a host cell that is a yeast. 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 accomplished with any species of yeast, including, but not limited to, any species of Yarrowia, such as, but not limited to, Yarrowia lipolytica. In some embodiments, the procedures and methods described herein can be accomplished with any species of yeast, including, but 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 are described in 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; Looke 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 of which is incorporated herein by reference in its entirety.

[0404] The yeast cell fermentation media and stock recipes are described 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 potassium phosphate monobasic, 5.17 g / L ammonium sulfate, 14.33 g / L potassium sulfate, 11.7 g / L magnesium sulfate heptahydrate, 2 mL / L PTM1 trace salts solution, 0.4 ppm biotin (500x from 200 ppm stock), 1-2% pure glycerol or other carbon source. (2) PTM1 trace salts solution: copper sulfate-5H2O 6.0 g, sodium iodide 0.08 g, manganese sulfate-H2O 3.0 g, sodium molybdate-2H2O 0.2 g, boric acid 0.02 g, cobalt chloride 0.5 g, zinc chloride 20.0 g, iron sulfate-7H2O 65.0 g, biotin 0.2 g, sulfuric acid 5.0 ml, add water to make a final volume of 1 liter. An exemplary composition of K. lactis defined 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, hydrogenated starch hydrolysate, 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, gas-permeable cover after inoculation). A colony of yeast (e.g., K. lactis) cultured on the plate can be picked and inoculated into a deep-well plate containing 2.2 mL of medium (composed of DMS or DMSO) per well. The inoculated deep-well plate can be grown for 6 days at 23.5°C with shaking at 280 rpm in a refrigerated incubator shaker. On the 6th day after inoculation, the conditioned medium should be collected by centrifugation at 4000 rpm for 10 minutes. It is then filtered using a filtration plate equipped 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 for yeast transformation is as follows: an expression vector carrying the CRIP ORF, CRIP-insecticide protein ORF, or Peptide-IA ORF is transformed into yeast cells. First, the expression vector is linearized, typically by specific restriction enzyme cleavage to facilitate integration into the chromosome via homologous recombination. The linear expression vector is then transformed into yeast cells by chemical or electroporation methods of transformation and integrated into the target locus in the yeast genome by homologous recombination. Integration can occur multiple times at the same chromosomal locus. Thus, the genome of transformed yeast cells can contain multiple copies of the CRIP or Peptide-IA expression cassette. Successfully transformed yeast cells can be identified using growth conditions that favor a selectable marker engineered into the expression vector and co-integrated into the yeast chromosome with the CRIP, CRIP-insecticide protein, or Peptide-IA ORF. Examples of such markers include, but are not limited to, acetamide prototrophy, zeocin resistance, geneticin resistance, nourseothricin resistance, and uracil prototrophy.

[0407] Due to the influence of unpredictable and variable factors (e.g., epigenetic modifications of genes and gene networks and variability in the number of integration events occurring in individual cells of a population undergoing a transformation procedure), individual yeast colonies of a given transformation process will differ in their ability to produce the CRIP ORF, the CRIP-insecticidal protein ORF, or the peptide-IA ORF. Therefore, transgenic yeast colonies carrying the CRIP or peptide-IA transgene should be screened for high-yielding strains. Two effective methods for such screening (each relying on the growth of small-scale cultures of transgenic yeast that provide conditioned medium samples for subsequent analysis) use reverse-phase HPLC or the housefly injection procedure to analyze conditioned medium samples from positive transgenic yeast colonies.

[0408] Transgenic yeast cultures can be performed using 14 mL round-bottom polypropylene culture tubes with 5–10 mL of defined medium added to each tube, or in 48-well deep-well culture plates with 2.2 mL of defined medium added to each well. Defined medium without crude proteinaceous extracts or by-products (e.g., yeast extract or peptone) is used for culture to reduce protein background in the conditioned medium recovered for subsequent screening steps. Cultures are carried out at optimal temperatures (e.g., 23.5 °C for K. lactis) for approximately 5–6 days until 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, and the supernatant is collected as conditioned medium. It is then clarified 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 analytical HPLC column with a C18 bonded phase. Acetonitrile and water are used as the mobile phase solvents, and a UV absorbance detector set at 220 nm is used for peptide detection. An appropriate amount of conditioned medium sample is loaded onto the rpHPLC system and eluted with a linear gradient of the mobile phase solvent. The corresponding peak area of ​​the insecticidal peptide in the HPLC chromatograph is used to quantify the CRIP or peptide-IA concentration in the conditioned medium. 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: The 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 integrative expression vector. Once the CRIP or peptide-IA transgene was cloned into pKLAC1 and transformed into YCT306, its expression was controlled by the LAC4 promoter. The resulting transformed colonies produced a pre-pro-peptide containing the α-mating factor signal peptide, a Kex2 cleavage site, and mature CRIP or peptide-IA. The α-mating factor signal peptide directs the pre-pro-peptide to enter the endogenous secretory pathway, and mature CRIP or peptide-IA is released into the growth medium.

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

[0412] In some embodiments, the yeast Pichia pastoris can be transformed with a CRIP, CRIP-insecticidal protein, or peptide-IA expression cassette. An exemplary method for transforming P. pastoris is as follows: CRIP or peptide-IA can be transformed into P. pastoris using the vectors pJUGαKR and pJUZαKR. The pJUGαKR and pJUZαKR vectors are available from Biogrammatics, Carlsbad, California, USA. Both vectors are integrating vectors and use the uracil phosphoribosyltransferase promoter (pUPP) to enhance expression of heterologous transgenes. The only difference between the vectors is that pJUGαKR confers G418 resistance to the host yeast, while pJUZαKR confers zeocin resistance. Pairs of complementary oligonucleotides encoding CRIP or peptide-IA are designed and synthesized for subcloning into the two yeast expression vectors. Hybridization reactions are performed by mixing the corresponding complementary oligonucleotides to a final concentration of 20 μM in 30 mM NaCl, 10 mM Tris-Cl (pH 8) (all final concentrations) and then incubating at 95°C for 20 minutes, followed by a 9-hour incubation period, 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. The two P. pastoris vectors are digested with BsaI-HF restriction enzymes, and the double-stranded DNA product of this reaction is then subcloned into a linearized P. pastoris vector using standard procedures. After sequence verification of the subclone, an aliquot of the plasmid is transfected by electroporation into P. pastoris strain Bg08. The resulting transformed yeast are selected based on resistance to Zeocin or G418 conferred by the engineered elements to the vectors pJUZαKR and pJUGαKR, respectively, and can be cultured and screened as described herein.

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

[0414] Screening and evaluation of yeast peptide yields Peptide yield can be determined by any of the methods known to those of skill 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, these or any other methods known in the art can be used to assess peptide yield.

[0415] Quantification assay In some embodiments, CRIP peptide yield can be measured using, but is not limited to, the following: HPLC, mass spectrometry (MS) and related techniques, LC / MS / MS, reverse phase protein array (RPPA), immunohistochemistry, ELISA, suspension bead array, mass spectrometry, dot blot, SDS-PAGE, capillary gel electrophoresis (CGE), Western blot analysis, Bradford assay, measuring UV absorbance at 260 nm, Lowry assay, Smith copper / bicinchoninic acid assay, secretion assay, Pierce protein assay, biuret reaction, etc. Exemplary methods for protein quantification include those described in 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, U.S. Patent No. 6,391,649 (the disclosures of which are incorporated herein by reference in their entireties).

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

[0417] In some embodiments, yields are expressed in terms of culture volume, and culture cell density can be taken into account, especially when yields between different cultures are being compared.

[0418] In some embodiments, the present invention provides methods for producing heterologous polypeptides that are 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 the total cellular protein (tcp). "Percentage of total cellular protein" is the amount of heterologous polypeptide in a host cell as a percentage of aggregated cellular protein. Determining percent total cellular protein is well known in the art.

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

[0420] In some embodiments, HPLC can be used to quantify peptide yield. For example, in some embodiments, the yield of CRIP or Peptide-IA can be assessed using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm C18 reverse-phase analytical HPLC column and an autoinjector. An exemplary use of an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm C18 reverse-phase analytical HPLC column and an autoinjector is as follows: Filtered conditioned medium samples from transformed K. lactis cells are analyzed using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm C18 reverse-phase analytical HPLC column and an autoinjector, with HPLC-grade water and acetonitrile containing 0.1% trifluoroacetic acid comprising the two mobile phase solvents used in the HPLC analysis. The peak areas of both CRIP and peptide-IA were analyzed using HPLC chromatography and then used to calculate the peptide concentration in the conditioned medium, which can be further normalized to the corresponding final cell density (determined by measuring OD600) 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 measured doses. The efficacy of CRIP or Peptide-IA is measured by the median paralytic / lethal dose (PD) of the peptide. 50 / LD 50 ) which results in a 50% knockdown rate or mortality rate of injected houseflies, respectively. Pure CRIP or peptide-IA are typically used in housefly injection assays, and PD 50 / LD 50 Generate standard dose-response curves from which values ​​can be determined. PD from analysis of standard dose-response curves of pure CRIP or peptide-IA.50 / LD 50 Using these values, quantification of CRIP or peptide-IA produced by the transformed yeast can be achieved using a housefly injection assay (performed with serial dilutions of the corresponding conditioned medium).

[0422] An exemplary housefly injection bioassay is as follows: Serial dilutions of conditioned medium are made to generate a complete dose-response curve from the housefly injection bioassay. Prior to injection, adult houseflies (Musca domestica) are immobilized with CO2 and 12–18 mg of flies are selected for injection. Using a microapplicator equipped with a 1 cc syringe and a 30-gauge needle, 0.5 μL of serially diluted conditioned medium sample per fly is injected into the housefly through the body wall of the dorsal thorax. Injected houseflies are placed in a sealed container with a moistened filter paper and a breathing hole on the lid, and they are examined 24 h after injection by scoring for knockdown rate or mortality. A normalized yield is calculated. Peptide yield refers to the peptide concentration in mg / L in the conditioned 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 cultures are harvested, different cultures may have different cell densities. Even if a strain's peptide production rate is lower 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-yielding 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 CRIP or Peptide-IA of at least up to 4 g / L, or at least up to 3 g / L, or at least up to 2 g / L can be achieved. Higher production rates (expressed in mg / L) may be from about 100 mg / L to about 100,000 mg / L, or from about 100 mg / L to about 90,000 mg / L, or from about 100 mg / L to about 80,000 mg / L, or from about 100 mg / L to about 70,000 mg / L, or from about 100 mg / L to about 60,000 mg / L, or from about 100 mg / L to about 50,000 mg / L, or from about 100 mg / L to about 40,000 mg / L, or from about 100 mg / L to about 30,000 mg / L, or about 100 mg / L to about 20,000 mg / L, or about 100 mg / L to about 17,500 mg / L, or about 100 mg / L to about 15,000 mg / L, or about 100 mg / L to about 12,500 mg / L, or about 100 mg / L to about 10,000 mg / L, or about 100 mg / L to about 9,000 mg / L, or about 100 mg / L to about 8,000 mg / L, or about 100 mg / L to about 7,000 mg / L, or about 100 mg / L to about 6,000 mg / L, or about 100 mg / L to about 5,000 mg / L, or about 100 mg / L to about 3,000 mg / L, or about 100 mg / L to 2,000 mg / L, or about 100 mg / L to 1,500 mg / L, or about 100 mg / L to 1,000 mg / L, or about 100 mg / L to 750 mg / L, or about 100 mg / L to 500 mg / L, or about 150 mg g / L to 100,000 mg / L, or about 200 mg / L to 100,000 mg / L, or about 300 mg / L to 100,000 mg / L, or about 400 mg / L to 100,000 mg / L, or about 500 mg / L to 100,000 mg / L, or about 750 mg / L to 100,000 mg / L, or about 1,000 mg / L to 100,000 mg / L, or about 1,250 mg / L to 100,000 mg / L, or about 1,500mg / L to 100,000mg / L, or about 2,000mg / L to 100,000mg / L, or about 2,500mg / L to 100,000mg / L, or about 3,000mg / L to 100,000mg / L, or about 3,500mg / L to 100,000mg / L, or about 4,000mg / L to 100,000mg / L, or about 4,500mg / L to 100,000mg / L, or about 5,000mg / L to 100,000mg / L, or about 6,000mg / L to 100,000mg / L, or about 7,000mg / L to 100,000mg / L, or about 8,000mg / L to 100,000mg / L, or about 9,000mg / L to 100,000mg / L, or about 10,000mg / L to 100,000mg / L, or about 12,500mg / L to 100,000mg / L, or or about 15,000mg / L to 100,000mg / L, or about 17,500mg / L to 100,000mg / L, or about 20,000mg / L to 100,000mg / L, or about 30,000mg / L to 100,000mg / L, or about 40,000mg / L to 100,000mg / L, or about 50,000mg / L to 100,000mg / L, or about 60,000mg / L to 100, The yield may be about 10,000 mg / L, or about 70,000 mg / L to 100,000 mg / L, or about 80,000 mg / L to 100,000 mg / L, or about 90,000 mg / L to 100,000 mg / L, or any range of any of the values ​​provided, or a yield even higher than that which could be achieved with the peptide before conversion using the same or similar production methods used to produce the peptide before conversion.

[0424] Any of the aforementioned methods can be used and / or adapted to produce CRIP and / or peptide-IA (e.g., a pesticide, e.g., a polymer of amino acids, peptides, and / or proteins, that is subjected to such a method). For example, any of the aforementioned methods can be used to produce, generate, produce, express, transcribe, translate, synthesize, or otherwise generate 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-Talb, TVP, Av2, Av3, AVP, and / or Bt toxin (e.g., Cry toxin, Cyt toxin, or Vip).

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

[0426] Exemplary culture methods are provided in U.S. Pat. Nos. 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, the disclosures of which are incorporated herein by reference in their entireties.

[0427] yeast culture Yeast cell culture techniques are well known to those skilled in the art. Exemplary methods for yeast cell culture are described in 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; Looke 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 of which 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 Synthetic Dropout Medium, Yeast Nitrogen Basal Medium (YNB, with or without amino acids), YEPD medium, ADE D medium, ADE DS medium, LEU D medium, HIS D medium, or mineral salts medium.

[0429] In some embodiments, the yeast may be cultured in a minimal medium, which 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 includes bacteriological peptone, yeast extract, and glucose.

[0431] In some embodiments, yeast can be cultured on yeast synthetic dropout medium, which can be used to distinguish auxotrophic mutants unable to grow without a particular medium component that have been transformed with a plasmid that allows the transformant to grow on medium lacking the required component.

[0432] In some embodiments, yeast can be cultured using Yeast Nitrogen Source Basal Medium (YNB, with or without amino acids), which contains nitrogen, vitamins, trace elements, and salts.

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

[0434] In some embodiments, the medium may be A DE D medium, e.g., a medium comprising 0.056% Ade-Trp-Thr powder, 0.67% yeast nitrogen source basal medium without amino acids, 2% D-glucose, and 0.5% 200x 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 medium may be ADE DS" medium, e.g., a medium containing 0.056% Ade-Trp-Thr powder, 0.67% yeast nitrogen source basal medium without amino acids, 2% D-glucose, 0.5% 200x tryptophan threonine solution, and 18.22% D-sorbitol, or a variation thereof, in which the carbon source is entirely a sugar alcohol (e.g., glycerol or sorbitol).

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

[0438] In some embodiments, a mineral salts medium can be used. Mineral salts medium consists of a mineral salt and a carbon source (e.g., glucose, sucrose, or glycerol). Examples of mineral salts medium include M9 medium, Pseudomonas aeruginosa (ATCC 179), and Davis-Mingioli medium. See Davis & Mingioli (1950) J. Bact. 60:17-28. Mineral salts used to prepare mineral salts medium include, for example, potassium phosphate, ammonium sulfate or chloride, magnesium sulfate or 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 extract are not included in mineral salts medium. Instead, an inorganic nitrogen source is used, which can be selected from, for example, ammonium salts, aqueous ammonia, and gaseous ammonia. Mineral salts medium typically contains glucose or glycerol as a carbon source.

[0439] In comparison to mineral salts media, minimal media can also contain mineral salts and a carbon source, but can be supplemented with, for example, low levels of amino acids, vitamins, peptones, or other components (although these are added at very minimal levels). The media can be prepared using methods described in the art, for example, in U.S. Patent Application Publication No. 2006 / 0040352, the disclosure of which is incorporated herein by reference in its entirety. Details of the culture procedure and mineral salts media useful in the methods of the present invention are described by 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, and cultures are incubated at 30°C until mid-log phase (24-48 hours) for β-galactosidase measurements, or at 23.5°C for 6 days for heterologous protein expression.

[0441] In some embodiments, yeast cells can be cultured in 48-well deep-well plates (sealed with a sterile, gas-permeable cover after inoculation). A colony of yeast (e.g., K. lactis) cultured on the plate can be picked and inoculated into a deep-well plate containing 2.2 mL of medium (composed of DMS or) per well. The inoculated deep-well plate can be grown for 6 days at 23.5°C, shaking at 280 rpm in a refrigerated incubator shaker. On the 6th day after inoculation, the conditioned medium should be collected by centrifugation at 4000 rpm for 10 minutes. Subsequently, it is filtered using a filtration plate equipped 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 modified using the methods described herein.

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

[0444] The pH level is important in yeast cultivation. Those skilled in the art will understand that the cultivation process includes not only the initiation of yeast culture but also the maintenance of the culture. Yeast culture can be initiated at any pH level, but care must be taken to monitor the pH level during the cultivation process, as the yeast 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 required pH level based on the yeast species used, the stage of culture, and / or the temperature. Thus, in some embodiments, the pH level may fall within a range of about 2 to about 10. Those skilled in the art will recognize that the optimal pH for most microorganisms is near neutral (pH 7.0). However, in some embodiments, some fungal species prefer an acidic environment. Thus, in some embodiments, the pH may range from about 2 to about 6.5. In some embodiments, the pH may range from 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 about 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 medium can be at least 5.5. In other aspects, the medium can have a pH level of about 5.5. In other aspects, the medium can have a pH level of 4 to 8. Optionally, the culture is maintained at a pH level of 5.5 to 8. In other aspects, the medium has a pH level of 6 to 8. Optionally, the medium has a pH level 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 about 5.6, 5.7, 5.8, or 5.9. In some embodiments, the yeast is grown at a pH level of about 6. In some embodiments, the yeast is grown at a pH level of about 6.5. In some embodiments, the yeast is grown at a pH level of about 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 about 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 pH level greater than 8.

[0448] In some embodiments, the pH of the medium can range from pH 2 to 8.5. In certain embodiments, the pH is about 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 culturing yeast can be found in U.S. Pat. No. 5,436,136, entitled "Repressible Yeast Promoters," filed 12 / 20 / 1991, assigned to Ciba-Geigy Corporation; U.S. Pat. No. 6,645,739, entitled "Yeast Expression Systems, Methods for Producing Polypeptides in Yeast, and Related Compositions," filed 07 / 26 / 2001, assigned to Phoenix Pharmacologies, Inc., Lexington, KY; and U.S. Pat. No. 10,023,836, entitled "Culture Media for Yeast," filed 08 / 23 / 2013, assigned to Yamaguchi University, the disclosures of which are incorporated herein by reference in their entireties.

[0450] fermentation The present invention contemplates culturing the host organism in any fermentation format, for example, batch, fed-batch, semi-continuous, and continuous fermentation modes can be used herein.

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

[0452] In some embodiments, the fermentation volume is about 1 liter or more. For example, in some embodiments, the fermentation volume is about 1 liter to about 100 liters. In some embodiments, the fermentation volume is about 1 liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters. In some embodiments, the fermentation volume is about 1 liter to about 5 liters, about 1 liter to about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 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 to grow and / or maintain cells. Without limitation, 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 a basis 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 range)).

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

[0455] In some embodiments, the fermentation medium can 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) protein and tissue hydrolysates (e.g., peptones or peptone mixtures that can be obtained from purified gelatin, plant material, or animal by-products), (6) antibiotics (e.g., gentamicin), and (7) cytoprotectants (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. Control of pH 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 being used. Thus, in some embodiments, the pH can range from about 1 to about 10, depending on the host cell and temperature.

[0457] In some embodiments, the pH of the fermentation medium can range from a pH of 2 to 8.5. In particular embodiments, the pH is about 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 about 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 temperature.

[0460] In other embodiments, for example, when yeast strains are used, the pH may range from about 4.0 to 8.0.

[0461] In some embodiments, a neutral pH (ie, 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 production of substrates and metabolic compounds.

[0463] In some embodiments, the fermentation medium can be supplemented with buffers or other chemicals to avoid changes in pH. For example, in some embodiments, additions of Ca(OH), CaCO, NaOH, or NHOH can be added to the fermentation medium to neutralize the production of acidic compounds that occur in some yeast species during industrial processes, for example.

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

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

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

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

[0468] Fermentation can be accomplished with a variety of microorganisms known to those skilled in the art. Microorganisms suitable for 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 strains of Saccharomyces species (including, but not limited to, S. cerevisiae (baker's yeast), S. distaticus, and S. uvarum), Kluyveromyces (including, but not limited to, K. marxianus and K. fragilis), Candida (including, but not limited to, C. pseudotropicalis and C. brassicae), Pichia stipitis (a relative of Candida shehatae), Clavispora (including, but not limited to, C. lusitaniae and C. opuntiae), Pachysolen (including, but not limited to, P. tannophilus), and Brettanomyces (e.g., 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., Pseudozyma tsukubaensis, yeast species (Zygosaccharomyces, Debaryomyces, Hansenula, and Pichia genera), and fungi of the dematioid genus Torula. See, e.g., 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 cell and / or the end user. Any necessary supplements other than carbon may also be included at appropriate concentrations, introduced alone or in mixtures with other supplements or media, such as complex nitrogen sources.

[0470] yeast fermentation Fermentation methods using yeast are well known to those skilled in the art. In some embodiments, batch fermentation can be used in accordance with the methods provided herein, while in other embodiments, continuous fermentation procedures can be used.

[0471] In some embodiments, a batch method of fermentation can be u...

Claims

1. A combination comprising a cysteine-rich insecticidal peptide (CRIP) and an insecticide (IA), wherein the CRIP is a U1-agatoxin-Ta1b variant polypeptide (TVP) having an amino acid sequence selected from any one of SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654, and the IA is a Bt toxin protein isolated from Bacillus thuringiensis var. kurstaki (Btk).

2. 2. The combination of claim 1, wherein the Bt toxin protein is one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk).

3. 3. The combination of claim 2, wherein the Bt toxin protein is one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis ssp. kurstaki strain EVB-113-19.

4. 4. The combination of claim 1, wherein the ratio of IA to CRIP is about 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.

5. The combination of claim 4, wherein the ratio of one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to CRIP is about 1:1 to about 1:

10.

6. 6. The combination of claim 5, wherein the ratio of one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to CRIP is about 1:9.

2.

7. The combination of claim 4, wherein the ratio of one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to CRIP is from about 1:1 to about 1:1.

5.

8. 8. The combination of claim 7, wherein the ratio of one or more fermentation solids, spores, or toxins isolated from Bacillus thuringiensis var. kurstaki (Btk) to CRIP is about 1:1.

375.

9. A combination 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 according to the amino acid sequence set forth in SEQ ID NO:

2.

10. A method of using a combination according to any one of claims 1 to 9 to control insects, comprising applying the combination according to any one of claims 1 to 9 to the site of the insect.

11. 11. The method of claim 10, 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), Armyworm (Spodoptera spp. (e.g., exigua, frugiperda, littoralis), Pseudaletia unipuncta), Artichoke Plume Moth (Platyptilia carduidactyla), Azalea Caterpillar (Datana major), Bagworm (Thyridopteryx ephemeraeformis), Banana Moth (Hypercompe scribonia), Banana Skipper (Erionota thrax), Blackheaded Budworm (Acleris gloverana), California Oakworm (Phyryganidia californica), Spring Cankerworm (Paleacrita merriccata), Cherry Fruitworm (Grapholita packardi), China Mark Moth (Nymphula stagnata), Citrus Cutworm (Xylomyges curialis), Codling Moth (Cydia pomomonella), Cranberry Fruitworm (Acrobasisvaccinii), Cross-striped Cabbageworm (Evergestis rimosalis), Cutworm (Noctuid species, Agrotis ipsilon), Douglas Fir Tussock Moth (Orgyia pseudotsugata), Ello Moth (Hornworm) (Erinnyis ello), Elm Spanworm (Ennomos subsignaria), European Grapevine Moth (Ernomos spp.), Grapevine Moth (Lobesia botrana), European Skipper (Thymelicus lineola (Essex Skipper)), Fall Webworm (Melissopus latiferreanus), Filbert Leafroller (Archips rosanus), Fruittree Leafroller (Archips argyrospiria), Grape Berry Moth (Paralobesia viteana), Grape Leafroller (Grape Leafroller (Platynota sultana), Grapeleaf Skeletonizer (Harrisina americana (ground only)), Green Cloverworm (Platypena scabra), Greenstriped Mapleworm (Dryocampa rubicunda, Gummosos-Batrachedra, Comosae (Hodges)), Gypsy Moth (Lymantria dispar), Hemlock Looper (Lambdaria fiscellaria), Hornworm (Manducaspp.), Imported Cabbageworm (Pieris rapae), Io Moth (Automeris io), Jack Pine Budworm (Choristoneura pinus), Light Brown Apple Moth (Epiphyas postvittana), Melonworm (Diaphania hyalinata), Mimosa Webworm (Homadauula anisocentra), Oblique Banded Leafroller (Choristoneura rosaceana), Oleander Moth (Syntomeiida epilais), Omnivorous Leafroller (Playnota sultana), Omnivorous Looper (Saboulodes aegrotata), Orange Dog (Papilio cresphontes), 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 (Schizuura concinna), Rindworm Complex (Various Leps.), Saddleback Caterpillar (Sibine stimulea)), Saddle Prominent Caterpillar (Heterocampa guttivitta), Saltmarsh Caterpillar (Estigmene acreaa), Sod Webworm (Crambus spp.), Spanworm (Ennomos subsignaria), Fall Cankerworm (Alsophila pometaria), Spruce Budworm (Choristoneura fumiferana), Tent Caterpillar (Various Lasiocampidae), Thecla-Thecla Basilides (Geyr) Thecla basilides), Tobacco Hornworm (Manduca sexta), Tobacco Moth (Ephestia elutella), Tufted Apple Budmoth (Platynota idaeusalis), Twig Borer (Anarsia lineatella), Variegated Cutworm (Peridroma saucia), Variegated Leafroller (Platynota flavedana), Velvetbean Caterpillar (Anticarsia gemmatalis), Walnut Caterpillar (Datana integerma), Webworm (Hyphantria cunea), Western Tussock Moth (Orgyia vetusta), Southern Cornstalk Borer (Diatraea crambidoides), Corn Earworm (CornEarworm, Sweet Potato Weevil, Pepper Weevil, Citrus Root Weevil, Strawberry Root Weevil, Pecan Weevil, Filbert Weevil, Ricewater Weevil, Alfalfa Weevil, Clover Weevil, Tea Shot-Hole Borer, Root Weevil, Sugarcane Beetle beetle), coffee berry borer, annual bluegrass weevil (Listronotus maculicollis), Asiatic garden beetle (Maladera castanea), European chafer (Rhizotroquus 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 suppressalis, Culex pipiens, CulexQuinquefasciatus, Diabrotica virgifera, Diatraea saccharalis, Helicoverpa armigera, Helicoverpa zea, Heliothis virescens, Leptinotarsa ​​decemlineata, Ostrinia furnacalis, Ostrinia nubilalis, Pectinophora gos sypiella, Plodia interpunctella, Plutella xylostella, Pseudoplusia includens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Trichoplusia ni, and Xanthogaleruca luteola.

12. A method of using a combination according to any one of claims 1 to 9 to control Bacillus thuringiensis toxin-resistant insects, comprising providing a combination according to any one of claims 1 to 9 and then applying the combination to the site of the insect.

13. 13. The method of claim 12, wherein the Bacillus thuringiensis resistant toxin insect is selected from the group consisting of: 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.

14. 10. A method of combating, controlling or suppressing pests, comprising applying a pesticidally effective amount of the combination of any one of claims 1 to 9 to the site of the pest or to a plant or animal susceptible to attack by the pest.

15. 15. The method of claim 14, wherein the pest is selected from the group consisting of: ahimas phynchus moth (hornworm) (Eumorpha achemon), alfalfa caterpillar (Colias eurytheme), almond moth (Caudra cautella), amorbia moth (Amorbia humerosana), armyworms (Spodoptera spp. (e.g., exigua, frugiperda, littoralis), Pseudaletia unipuncta), artichoke plume moth (Platyptia carduidactyla), azalea caterpillar (Datana major), bagworm (Thyridopteryx), ephemeraeformis, banana moth (Hypercompe scribonia), banana skipper (Erionota thrax), black-headed budworm (Acleris gloverana), California oakworm (Phyryganidia 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 (Lobesia botrana), European skipper (Thymelicus lineola (Essex skipper)), fall webworm (Melissopus latiferreanus), filbert leaf roller (Archips rosanus, fruit tree leaf roller (Archipsargyrospiria, grapeberry moss (Paralobesia viteana), grape leaf roller (Platynota sultana), grape leaf skeletonizer (Harrisina americana (ground only)), green clover worm (Platypena scabra), green striped maple worm (Dryocampa rubicunda, Gummosos-Batrachedra, Comosae (Hodges)), gypsy moss (Lymantria dispar), hemlock looper (Lambdaria fiscellaria), hornworm (Manduca spp.), imported cabbage worm (Pieris rapae), Io moss (Automeris io), Jack pine budworm (Choristoneura pinus), Light brown apple moss (Epiphyas postvittana), Melon worm (Diaphania hyalinata), Mimosa webworm (Homadauula anisocentra), Oblique banded leafroller (Choristoneura rosaceana), Oleander moss (Syntomeiida epilais), Omnivorous leafroller (Playnota sultana), Omnivorous looper (Saboulodes aegrotata), Orange dog (Papilio cresphontes), Orange Tortrix (Argyrotaenia citrana), Oriental Fruit Moth (Grapholita molesta), Peach Twigboa (Anarsia lineatella), Pine Butterfly (Neophasia menapia), Podworm (Heliocoverpa zea), Red Banded Leaf Roller (Argyrotaenia velutinana), Red Hump Caterpillar (Schizura concinna), Lindworm Complex (Various Leps.), Saddleback Caterpillar (Sibine stimulea), Saddle Prominent Caterpillar (Heterocampa guttivitta), Salt Marsh Caterpillar (Estigmeneacrea), sodbedworm (Crambus spp.), spanworm (Ennomos subsignaria), fallcankerworm (Alsophila pometaria), sprucebudworm (Choristoneura fumiferana), tentcaterpillar (Various Lasiocampidae), Thecla-Thecla basilides (Geyr) Thecla basilides), tobaccohornworm (Manduca sexta), tobaccomoth (Ephestia elutella), tufted applebudmoth (Platynota idaeusalis), twigboer (Anarsia lineatella), bare-eared gated cutworm (Peridroma saucia), bare-eared gated leaf roller (Platynota flavedana), velvet bean caterpillar (Anticarsia gemmatalis), walnut caterpillar (Datana integerma), webworm (Hyphantria cunea), western tussock moss (Orgyia vetusta), southern corn stalk boar (Diatrea crambidoides), Corn Earworm, Sweet Potato Weevil, Pepper Weevil, Citrus Root Weevil, Strawberry Weevil, Pecan Weevil, Filbert Weevil, Ricewater Weevil, Alfalfa Weevil, Clover Weevil, Tee-Hole Borer, Root Weevil, Sugarcane Beetle, Coffeeberry Borer, Annual Bluegrass Weevil (Listronotus maculicollis), Asiatic Garden Beetle (Maladera castanea), European Chafer (Rhizotroquus majalis), Green June Beetle (Cotinis nitida), Japanese Beetle (Popillia japonica), Mayo June Beetle (Phyllophaga sp.), Northern Masked Chafer (Cyclocephala borealis), Oriental Beetle (Anomalaorientalis), 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.

16. 16. The method of claim 15, wherein the pest is selected from the group consisting of: Aedes aegypti, Busseola fusca, Chilo suppressalis, Culex pipiens, Culex quinquefasciatus, Diabrotica virgina, 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.

17. A combination described in any one of claims 1 to 9, wherein the combination is formulated into separate compositions or the same composition, and each of the separate compositions or the same composition further comprises one or more excipients.

18. The combination of claim 17, wherein the separate compositions or the same composition are formulated as a powder, dust, pellets, granules, spray, emulsion, colloid, solution or combinations thereof.

19. The combination of claim 18, wherein the separate compositions are formulated using the same excipients or different excipients.

20. A combination described in any one of claims 1 to 9, wherein: (1) an insecticide (IA) is insertionally expressed in a plant and a cysteine-rich insecticidal peptide (CRIP) is formulated as a composition comprising one or more excipients; (2) CRIP is insertionally expressed in a plant and IA is formulated as a composition comprising one or more excipients; or (3) both IA and CRIP are insertionally expressed in a plant.

21. A combination comprising a cysteine-rich insecticidal peptide (CRIP), an insecticide (IA), and an excipient, wherein the CRIP is a U1-agatoxin-Ta1b variant polypeptide (TVP) having an amino acid sequence selected from any one of SEQ ID NOs: 2-15, 49-53, 621-622, 624-628, 631-640, 642-651, or 653-654, and the IA is a Bt toxin protein isolated from Bacillus thuringiensis var. kurstaki (Btk).

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