U1-agatoxin-TA1B variant polypeptide for pest control, stable against protein degradation.

Abstract

Novel insecticidal proteins, insecticidal nucleotides, and insecticidal peptides, and their expression in plants. Methods for producing the novel nucleotides and novel peptides, novel processes, novel production techniques, novel formulations, and novel organisms. This disclosure also relates to and describes a novel peptide called TVP, which is a non-naturally occurring peptide variant based on the U1-agatoxin-Ta1b toxin from the grass spider. Described herein are genes encoding TVP, formulations and combinations containing the TVP gene and / or peptide, and methods of use thereof useful for controlling insect pests.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of and priority under U.S. Provisional Application No. 63 / 012,755 filed April 20, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] array A sequence listing file titled "225312-490536_ST25.txt" (68KB), created at 6:39 p.m. on April 20, 2021, was submitted electronically with this specification, and the entire sequence listing is incorporated into this application by reference.

[0003] Novel insecticidal proteins, insecticidal nucleotides, insecticidal peptides, their expression in plants, methods for producing such peptides, novel processes, production techniques, novel peptides, novel formulations, and novel and known combinations of organisms that provide higher yields than expected with related peptides for controlling insects are described and requested. [Background technology]

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

[0005] Botflies and blind flies can transmit bacterial pathogens that cause tularemia (Pasteurella tularensis) and anthrax (Bacillus anthracis), as well as the parasitic roundworm (Loa loa) that causes loa filariasis in tropical Africa.

[0006] Black flies of the genus Hippelates can carry the spirochete pathogen (Treponema pertenue) that causes strawberry cysts and can 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 of the bacterium (Bartonella bacilliformis) that causes Carillon's disease (Oroya fever) in South America. In parts of Asia and North Africa, sand flies of the genus Phlebotomus spread the viral pathogen that causes sand fly fever (Papatasi fever) and the protozoan pathogen (Leishmania species) that causes leishmaniasis. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Provisional Application No. 63 / 012,755 [Overview of the project] [Means for solving the problem]

[0008] This disclosure provides U1-agatoxin-Ta1b variant polypeptide (TVP), compositions comprising TVP, insecticidal proteins comprising one or more TVPs optionally with other proteins, and methods of using the same to cause eradication, death, control, suppression, damage, disruption, sterilization, or a combination thereof of one or more insect species. The TVPs described herein have insecticidal activity against one or more insect species. The TVP of this disclosure has an amino acid sequence that is at least 95% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQK-X6, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, Q, 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; and X6 is G or absent. The TVP described herein is a concentration (KD) that knocks down 50% of a housefly population in 24 hours. 50 The saturation level was less than 200 pmol / g, and it has been shown that the mortality rate for thrips was 100% on day 4.

[0009] This disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide is U1-agatoxin-Ta1b as shown in SEQ ID NO: 1. Compared to the wild-type sequence a1b, it contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, Q, 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.

[0010] Furthermore, this disclosure describes a composition comprising a TVP and one or more pharmaceutical additives, wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, V, M, I, Q, 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 one or more pharmaceutical excipients are selected from the group consisting of trehalose, maltodextrin, maltose, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), lignosulfonates, gypsum, sorbitol, sodium benzoate, potassium sorbate, EDTA, benzisothiazolinone (BIT), and fermented solids.

[0011] Furthermore, this disclosure describes a composition comprising TVP and several pharmaceutical additives, wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, Q, C, E, T, or S, X3 is T or P, and 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, the amount of TVP in the composition constitutes 8.5% wt / wt of the total weight of the composition, and the multiple pharmaceutical additives consist of 25% wt / wt of trehalose, 0.05% wt / wt of BIT, 36.3% wt / wt of maltodextrin, 2.6% wt / wt of anhydrous dipotassium hydrogen phosphate (K2HPO4), 0.4% wt / wt of potassium dihydrogen phosphate (KH2PO4), and 26.85% wt / wt of fermented solids, relative to the total weight of the composition.

[0012] Furthermore, this disclosure describes a polynucleotide or complementary nucleotide sequence encoding a TVP, wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the TVP is the wild-type variant of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. Compared to the column, each contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, Q, 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.

[0013] Furthermore, this disclosure describes a method for generating a TVP, the method comprising (a) preparing a vector comprising a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding a TVP, wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises at least one amino acid sequence compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising an acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, Q, 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; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions that enable TVP expression and secretion into growth medium.

[0014] Furthermore, this disclosure describes a method for controlling insects using the above-described composition, the method comprising providing the composition to a location where the insects are present.

[0015] Furthermore, this disclosure describes a method for protecting plants from insects, which includes providing a plant expressing TVP or a polynucleotide encoding TVP.

[0016] Furthermore, this disclosure describes methods for controlling, eliminating, or suppressing pests, which include applying the above composition in an effective amount as a pesticide to a location where pests are present or to plants or animals susceptible to pest attack.

[0017] Furthermore, this disclosure describes a vector comprising a polynucleotide capable of functioning to encode a TVP having an amino acid sequence that is 90% similar to any one of the sequences shown in SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0018] Furthermore, this disclosure describes a yeast strain comprising a first expression cassette containing a polynucleotide or a complementary nucleotide sequence capable of encoding a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide is U1 as shown in SEQ ID NO: 1 Compared to the wild-type sequence of -agatoxin-Ta1b, it contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0019] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A.

[0020] Furthermore, this disclosure describes compositions comprising TVP or a pharmaceutically acceptable salt thereof and one or more pharmaceutical additives, wherein TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A, and the one or more pharmaceutical additives are selected from the group consisting of trehalose, maltodextrin, maltose, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), lignosulfonates, gypsum, sorbitol, sodium benzoate, potassium sorbate, EDTA, benzisothiazolinone (BIT), and fermented solids.

[0021] Furthermore, this disclosure describes a composition comprising TVP or a pharmaceutically acceptable salt thereof and a plurality of pharmaceutical additives, wherein TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and 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, Z1 is T or A, the amount of TVP in the composition constitutes 8.5% w / w of the total weight of the composition, and the multiple pharmaceutical additives consist of 25% w / w of trehalose, 0.05% w / w of BIT, 36.3% w / w of maltodextrin, 2.6% w / w of anhydrous dipotassium hydrogen phosphate (K2HPO4), 0.4% w / w of potassium dihydrogen phosphate (KH2PO4), and 26.85% w / w of fermented solids, relative to the total weight of the composition.

[0022] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs. 2, SEQ ID NOs. 49, or SEQ ID NOs. 51.

[0023] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP comprising the amino acid sequence shown in any one of SEQ ID NOs: 2, 49, or 51.

[0024] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP comprising the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 49, or SEQ ID NO: 51.

[0025] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 51.

[0026] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP comprising the amino acid sequence shown in SEQ ID NO: 51.

[0027] Furthermore, this disclosure describes an insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) or a pharmaceutically acceptable salt thereof, the TVP comprising the amino acid sequence shown in SEQ ID NO: 51. [Brief explanation of the drawing]

[0028] [Figure 1]The map shows a Ta1b embedding vector containing a 9210 base pair circular vector with the following sequences: bacterial Ori / AmpR, 3'Lac4 promoter, α-MF signal sequence (shown as "a-MF" in the figure), polynucleotide encoding the U1-agatoxin-Ta1b variant polypeptide (shown as "Ta1b" in the figure), Lac4 terminator, amdS marker (shown as "Amd marker"), and 5'Lac4 promoter. [Figure 2] The results of the housefly injection assay are shown. In this assay, active wild-type Ta1b, as well as TVP with a 9-position R→Q amino acid substitution and TVP with a 9-position R→N amino acid substitution, were injected into the thorax of houseflies, and the dose of TVP required for 50% knockdown (KD50) was determined 24 hours after injection. [Figure 3] The results of the thrips survival assay are shown. In this assay, the lethality (lethal %) of the untreated control (UTC), wild-type U1-agatoxin-Ta1b (Ta1bWT), and TVP with the R→Q amino acid substitution at position 9 was evaluated. [Figure 4] The HPLC chromatogram of TVP (TVP-R9Q) with a 9-position R→Q amino acid substitution is shown, with the putative glycosylated species indicated as a "shoulder" to the left of the major TVP peak (indicated by the black arrow). [Figure 5] The ESI-MS results are shown, and the mass spectrum of TVP-R9Q is presented. The inset shows that the calculated masses by deconvolution of the multivalent species are 5732 Da and 5894 Da. The predicted molecular weight of completely oxidized TVP-R9Q is 5731.3. [Figure 6] The HPLC chromatogram results for TVP-R9Q are shown. The smaller "shoulder" to the left of the main peak indicates glycosylation. [Figure 7] The HPLC chromatogram results for TVP-R9QΔG are shown. The smaller "shoulder" to the left of the main peak indicates glycosylation. The right shoulder indicates partial proteolysis. [Figure 8]The HPLC chromatogram results for TVP-R9Q / T43A / ΔG are shown. The presence of two shoulders demonstrates a proteolytic event. The smaller "shoulder" located to the left of the major peak indicates a partial proteolytic event. [Figure 9] The HPLC chromatogram results for TVP-R9Q / T43A are shown. A single species of TVP is present here. [Figure 10] The HPLC chromatogram results for TVP-R9Q / T43A / ΔK-G are shown. Here, a single species of TVP is present. [Figure 11] The ESI-MS results are shown, and the mass spectrum of TVP-R9Q / T43A / ΔG is presented. [Figure 12] The ESI-MS results are shown, and the mass spectrum of TVP-R9Q / T43A is presented. [Figure 13] The results of ESI-MS are shown, and the mass spectrum of TVP-R9Q / T43A / ΔK-G is presented. [Figure 14] The results of ESI-MS are shown, along with the mass spectrum of TVP-R9Q / T43A / ΔK-G. [Figure 15] The results of the housefly injection assay are shown. In this assay, TVP was used after the glycosylation site and / or C-terminal amino acid had been removed. [Figure 16] The results of stability assays evaluating liquid concentrate (LC) formulations over time at different temperatures are presented. The LC formulation, which contained 2% TVP-R9Q / T43A, 0.03% benzoisothiazolinone (BIT), 2% sorbitol, with the remainder being fermented beer, i.e., the fermented beer concentrate from which cells were isolated, was evaluated using HPLC to determine the stability of TVP-R9Q / T43A over time at different temperatures. The evaluation temperatures were 4°C, 21°C, 37°C, 45°C, and 54°C. The evaluation times were 0 days, 16 days, 31 days, 42 days, 98 days, and 114 days. [Figure 17]The results of a stability assay for TVP-R9Q / T43A in spray-dried powder (SDP) form are shown. Here, the SDP formulation was prepared from dried fermented beer. The stability of TVP-R9Q / T43A in SDP was evaluated using HPLC, quantifying the amount (mg / mL) of TVP-R9Q / T43A at 21°C, 37°C, and 45°C, with and without the use of oxygen absorber / desiccant packets. Results enclosed in black squares indicate those obtained with oxygen absorber / desiccant packets. [Figure 18] The results of evaluating the preliminary stability formulations by performing degradation assays are shown. The formulations were tested for two weeks at 54°C, 37°C, and 21°C. Each formulation number group (i.e., 7, 18, 21, 2-1, 2-2, and 2-3) is indicated by the formulation number, followed by a dash " / ", followed by the test temperature (i.e., 54°C, 37°C, and 21°C). [Figure 19] This report presents the results of stability assays performed on formulations containing benzoisothiazolinone (BIT) to determine their compatibility with TVP. Here, cell-removed, concentrated fermented beer obtained from cells expressing TVP-R9Q / T43A was used as the formulation. Samples were evaluated at time T0 (immediately after formulation and drying in a spray dryer (evaluation performed at room temperature)) and after 2 weeks of incubation at 54°C. Peptide concentrations were quantified by HPLC analysis as described herein. Each formulation was tested at 4°C, 21°C, 37°C, and 45°C. [Figure 20] This report presents the results of stability assays performed on formulations containing benzoisothiazolinone (BIT) to determine their compatibility with TVP. Here, spray-dried powder (SDP) containing TVP-R9Q / T43A was used as the formulation, and samples were evaluated at time T0 (immediately after compounding and drying in a spray dryer (evaluation performed at room temperature)) and after 2 weeks of incubation at 54°C. Peptide concentrations were quantified by HPLC analysis as described herein. Each formulation was tested at 4°C, 21°C, 37°C, and 45°C. [Figure 21]The results of the pH stability assay are shown. Here, pH stability screening was performed to determine the effect of pH on the stability of TVP-R9Q / T43A. TVP-R9Q / T43A was incubated at 1‰(ppt) different pH values. The percentage of residual peptide was determined by incubating TVP-R9Q / T43A at 45°C for 7 days and analyzing by HPLC. As shown here, the optimal pH was determined to be 6.5–7.5. [Figure 22] The results for Stability Panel Part I are shown. Part I includes formulation numbers 1-17. The test components include: TVP-R9Q / T43A in the form of liquid concentrate (LC) (i.e., concentrate of cell-isolated fermented beer), H2O (reverse osmosis), maltodextrin, trehalose, maltose, K2HPO4, KH2PO4, and BIT (a 9.25% aqueous solution of 1,2-benzoisothiazolin-3-one). Samples were evaluated at time T0 (immediately after formulation and drying in a spray dryer (evaluation performed at room temperature)) and after incubation at 54°C for 2 weeks. Peptide concentrations were quantified by HPLC analysis. [Figure 23] The results of Stability Panel Part II are shown. Part II includes formulation numbers 18-28. The test components include: TVP-R9Q / T43A in the form of liquid concentrate (LC) (i.e., concentrate of cell-isolated fermented beer), H2O (reverse osmosis), maltodextrin, trehalose, maltose, K2HPO4, KH2PO4, BIT (9.25% aqueous solution of 1,2-benzoisothiazolin-3-one), and lignosulfonates. The tests were evaluated at time T0 and after 2 weeks of incubation at 54°C. Peptide concentrations were quantified by HPLC analysis as described herein. [Figure 24] The image shows brittle solid aggregates formed during two weeks of storage at 54°C. [Figure 25] A photograph shows the physical morphology obtained after the addition of lignosulfonate. As shown here, although the addition of lignosulfonate prevented the formation of brittle solids, it did not prevent the aggregation that occurred after two weeks of incubation at 54°C. [Figure 26] The results of Stability Panel Part III are shown. Part III includes formulation numbers 18-28. The test components include: TVP-R9Q / T43A in the form of liquid concentrate (LC) (i.e., concentrate of cell-isolated fermented beer), H2O (reverse osmosis), trehalose, lignosulfonate, maltodextrin, K2HPO4, KH2PO4, and BIT (a 9.25% aqueous solution of 1,2-benzoisothiazolin-3-one). Here, the bar graph shows the theoretical peptide concentration (black bars) based on the composition design described in the table above. The actual peptide percentage at time T0 is shown by the gray bars. The actual peptide percentage after incubation at 54°C for 2 weeks is shown by the shaded bars. [Figure 27] A photograph shows the physical form of formulation number 35 (in the tray placed on the bag labeled "Form 7"). As shown here, formulation number 35 was able to maintain its dry, fluid powder form even after incubation at 54°C for two weeks. [Figure 28] A photograph shows the physical form of formulation number 36 (in the tray placed on the bag labeled "Form 8"). As shown here, formulation number 36 was able to maintain its dry, fluid powder form even after incubation at 54°C for two weeks. [Figure 29] This section outlines the theoretical formulations (average values) of four dry granular prototype formulations. [Figure 30] The results of testing four prototype samples (prototype numbers 1-4) using stability assays are shown. The high-temperature stability of the four prototypes was evaluated by incubation at 54°C for 2 weeks in the presence of oxygen absorber / desiccant packets. TVP-R9Q / T43A peptide measurements were performed using HPLC-UV at T0 (time after formulation and drying) and T2W (time after 2 weeks). [Figure 31] The image shows the physical morphology obtained from prototype number 1 after two weeks of incubation at 54°C. Here, the physical morphology is dry, fluid, granular. [Figure 32]The image shows the physical morphology obtained from prototype number 2 after two weeks of incubation at 54°C. Here, the physical morphology is dry, fluid, granular. [Figure 33] The image shows the physical morphology obtained from prototype number 3 after two weeks of incubation at 54°C. Here, the physical morphology is dry, fluid, granular. [Figure 34] The image shows the physical morphology obtained from prototype number 1 after two weeks of incubation at 54°C. Here, the physical morphology is dry, fluid, granular. [Figure 35] The results of the circular dichroism (CD) assay are shown. The addition of 10% trehalose stabilizes the tertiary structure of TVP-R9Q / T43A, as indicated by the sinking of the CD spectra at 214 nm and 220 nm. This sinking indicates that the peptide is undergoing alpha-helixization, which is consistent with the peptide's 3D structure. [Figure 36] The results of evaluating the melting characteristics using a circular dichroism (CD) assay are shown. Here, the melting point of TVP-R9Q / T43A increased from 71.6°C to 73.6°C by adding 10% trehalose, which supports the idea that the stability of TVP-R9Q / T43A is improved in the presence of trehalose. [Modes for carrying out the invention]

[0029] definition

[0030] The terms "5' end" and "3' end" refer to orientation, i.e., the orientation between the ends of a nucleotide polymer (e.g., DNA). The 5' end of a polynucleotide is the end of the polynucleotide that has the fifth carbon atom.

[0031] "5' homology arm and 3' homology arm," or "5' arm and 3' arm," or "left arm and right arm" refers to polynucleotide sequences in a vector and / or target-directed vector that induce homologous recombination with a target genome sequence and / or endogenous target gene in the host organism in order to successfully genetically modify the chromosomal locus of the host organism.

[0032] The "ADN1 promoter" refers to a DNA segment composed of a promoter sequence derived from the adhesion-deficient protein 1 gene of the Schizosaccharomyces pombe.

[0033] "To affect" refers to how something affects something else, for example, how a peptide, polypeptide, protein, drug, or chemical affects an insect (e.g., a pest).

[0034] "Agent" refers to one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, toxins, toxic substances, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryotes, or eukaryotes (and agents produced from such prokaryotes or eukaryotes).

[0035] "Agriculturally acceptable carriers" encompass all auxiliaries, inert components, dispersants, surfactants, tackifiers, binders, etc., commonly used in pesticide formulation technology, and these are well known to those skilled in the field of pesticide formulation.

[0036] In this specification, the term "agriculturally acceptable salt" is used synonymously with the term "medically acceptable salt."

[0037] "Agroinfection" refers to a plant transformation method in which DNA is introduced into plant cells using Agrobacteria tumefaciens or Agrobacteria rhizogenes.

[0038] "Alignment" refers to a method of comparing two or more sequences (e.g., nucleotide sequences, polynucleotide sequences, amino acid sequences, peptide sequences, polypeptide sequences, or protein sequences) with the aim of determining their relationship to each other. Alignment is typically performed by computer programs that apply various algorithms, but it can also be performed manually. Alignment programs typically employ various strategies to achieve a potential optimal alignment score by iterating through potential sequence alignments and scoring the alignments using a substitution table. 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).Examples of programs that perform one or more of the aforementioned algorithms include, but are not limited to, MegAlign (provided by DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705)), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio (provided by Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif. 92121)). In some embodiments, alignment involves introducing a "phase shift" and / or "gap" into one or both of the sequences being compared to maximize the similarity between the two sequences. Scoring refers to the process of quantitatively representing the relevance of the sequences being aligned.

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

[0040] "Arachnoids" refers to the class of arthropods. For example, in some embodiments, Arachnoids may mean spiders, scorpions, ticks, mites, harvestmen, or sun spiders.

[0041] "BAAS" stands for barley alpha-amylase signal peptide and is an example of an ERSP. An example of BAAS is the BAAS having the amino acid sequence of SEQ ID NO: 37 (NCBI acceptance number AAA32925.1).

[0042] "Biomass" refers to any plant product being measured.

[0043] A "binary vector" or "binary expression vector" refers to an expression vector that can replicate itself in both E. coli and Agrobacterium strains. This vector contains a DNA region (often called t-DNA) flanked by left and right boundary sequences, which is recognized by the pathogenic gene to be copied and delivered to plant cells by Agrobacterium.

[0044] "bp" or "base pair" refers to a molecule containing two chemical bases bonded together. For example, the DNA molecule consists of two helical strands, each with a backbone made up of alternating deoxyribose and phosphate groups. Each deoxyribose is to which one of four bases (i.e., adenine (A), cytosine (C), guanine (G), or thymine (T)) is attached, with adenine forming a base pair with thymine and cytosine forming a base pair with guanine.

[0045] The term "C-terminus" refers to the free carboxyl group (i.e., -COOH) located at the end of a polypeptide.

[0046] "cDNA," "copy DNA," or "complementary DNA" refers to a molecule complementary to an RNA molecule. 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 all nucleic acids that share the arrangement of sequence elements found in a naturally occurring mature mRNA species, such as exons and the 3' and 5' non-coding regions. Typically, mRNA species create a continuous open reading frame that codes for proteins by having adjacent exons interposed by introns that are removed by RNA splicing in the nucleus. In some embodiments, "cDNA" refers to DNA that is complementary to an mRNA template and is obtained from that mRNA template.

[0047] "CEW" refers to the larva of the tobacco budworm.

[0048] For information on "cuttable linkers," please refer to the linker section.

[0049] "Cloning" refers to the process and / or method of inserting a DNA segment (e.g., usually a target gene, e.g., tvp) from one source, causing recombination between this DNA segment and a DNA segment from another source (e.g., usually a vector, e.g., plasmid), and guiding the recombinant DNA, or "recombinant DNA," to replicate (usually by transforming the recombinant DNA into a bacterial or yeast host).

[0050] 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, under the control of appropriate regulatory sequences and in the presence of necessary transcriptional and / or translational molecular factors. The boundaries of a coding sequence are determined by a translation start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. The transcription termination sequence is usually located on the 3' side of the coding sequence. In some embodiments, the coding sequence may be adjacent to the 5' and / or 3' ends of an untranslated region. In some embodiments, the coding sequence may be used to produce a peptide product, polypeptide product, or protein product. In some embodiments, the coding sequence may or may not be fused with another coding sequence or localization signal (such as a nuclear localization signal). In some embodiments, the coding sequence may be cloned into a vector or expression construct, incorporated into a genome, or exist as a DNA fragment.

[0051] "Codon optimization" refers to generating a gene in which one or more endogenous, native, and / or wild-type codons are replaced with codons that ultimately still encode the same amino acids but have a preferred tendency in the corresponding host.

[0052] "Complementary," as understood by those skilled in the art, refers to the topological fit or match of the interaction surfaces of two polynucleotides. Thus, two sequences are "complementary" if they can hybridize with 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 with the second polynucleotide under strict hybridization conditions. Thus, a polynucleotide having the sequence 5'-TATAC-3' is complementary to a polynucleotide having the sequence 5'-GTATA-3'.

[0053] "Acclimatized medium" refers to a cell culture medium that is used by cells and has a high amount of cell-derived substances, but does not contain cells themselves.

[0054] "Copy number" refers to the number of identical copies of a vector, expression cassette, amplification unit, gene, or any specific nucleotide sequence present in a host cell at any given time. For example, in some embodiments, a gene or another specific chromosomal nucleotide sequence may have one, two, or more copies on a chromosome. A self-replicating vector may have hundreds or even hundreds of copies per host cell.

[0055] "Culture" or "cell culture" refers to maintaining cells in an artificial in vitro environment.

[0056] "Culturing" refers to growing organisms on or in various types of culture media. For example, the term "culturing" may mean growing a population of cells under appropriate conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentation-recombinant production of a desired heterologous polypeptide and / or other desired end product (typically in a container or reactor).

[0057] "Cystine" refers to an oxidized cysteine ​​dimer. Cystine is a sulfur-containing amino acid obtained by the oxidation of two cysteine ​​molecules, linked by a disulfide bond.

[0058] A "limited medium" refers to a medium that consists of known chemical components but does not contain unpurified protein extracts or by-products (such as yeast extract or peptone).

[0059] A "disulfide bond" refers to a covalent bond that forms between two cysteine ​​amino acids when two thiol groups on the side chains of those two cysteine ​​amino acids are coupled together.

[0060] "Degeneracy" or "codon degeneracy" refers to the phenomenon where a single amino acid can be coded by different nucleotide codons. Therefore, the nucleic acid sequences of nucleic acid molecules encoding proteins or polypeptides can differ due to degeneracy. As a result of genetic coding degeneracy, many nucleic acid sequences can code for a given polypeptide with specific activity. Such functionally equivalent variants are intended in this specification.

[0061] "DNA" refers to deoxyribonucleic acid, which comprises a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), and the polymer may exist in single-stranded or double-stranded form. For example, one or more nucleotides can create a polynucleotide.

[0062] "dNTP" refers to nucleoside triphosphates, which are components of DNA and RNA.

[0063] A "dual expression cassette" refers to two TVP expression cassettes contained within the same vector.

[0064] A "dual transgene peptide expression vector" or "dual transgene expression vector" refers to a yeast expression vector containing two copies of the TVP expression cassette.

[0065] "Endogenous" refers to a polynucleotide, peptide, polypeptide, protein, or process that occurs and / or exists naturally in an organism, for example, the molecule or activity already present in the host cell before any particular genetic manipulation is performed.

[0066] An "enhancer element" refers to a DNA sequence that is ligated to a promoter in a functional manner, and this DNA sequence can enhance the transcriptional activity of the promoter compared to the transcriptional activity produced by the promoter in the absence of the enhancer element.

[0067] The "ER," or "endoplasmic reticulum," is an intracellular organelle common to all eukaryotes, and some kind of post-translational modification process takes place within this organelle.

[0068] "ERSP," or "endoplasmic reticulum signal peptide," is the N-terminal amino acid sequence recognized and bound by a host cell signal recognition particle during the translation of the mRNA molecule encoding TVP into protein. This signal recognition particle moves the protein-translation ribosome / mRNA complex to the ER in the cytoplasm. As a result, protein translation is temporarily halted until this complex binds to the ER, at which point protein translation resumes and the resulting protein is injected into the ER.

[0069] "ersp" refers to the polynucleotide that codes for the ERSP peptide.

[0070] "ER transport" refers to the transport of cell-expressed proteins to the ER for post-translational modification, localization, and transport.

[0071] "Pharmaceutical additives" refers to any pharmacologically inactive natural or synthetic components or substances formulated together with (e.g., simultaneously with) or subsequently with the active ingredient of the present invention (i.e., TVP or TVP-insecticide protein). In some embodiments, pharmaceutical additives may be any additives, auxiliaries, binders, bulking agents, carriers, coatings, diluents, disintegrants, fillers, flow enhancers, lubricants, preservatives, media, or combinations thereof, together with the TVP or TVP-insecticide protein of the present invention, and / or such may be useful in the preparation of the compositions of the present invention. Pharmaceutical additives include any substances known in the art that are non-toxic and do not interact with other components of the composition. In some embodiments, pharmaceutical additives may be formulated together with TVP or TVP-insecticide protein at the time of preparation of the composition for the purpose of bulking the composition (and are therefore often referred to as bulking agents, fillers, or diluents). In other embodiments, pharmaceutical additives may be used to enhance (e.g., enhance absorption and / or solubility) the active ingredient in the final dosage form. In yet another embodiment, pharmaceutical additives may be used to impart stability or prevent contamination (e.g., microbial contamination). In yet another embodiment, pharmaceutical additives may be used to impart physical properties to a composition (e.g., a composition in dry granular physical form or dry flowable powder physical form). References to pharmaceutical additives include both one such pharmaceutical additive and multiple such pharmaceutical additives. Suitable pharmaceutical additives are described in Remington's Pharmaceutical Sciences by E.W. Martin, and the disclosures of said document are incorporated herein by reference in their entirety.

[0072] An "expression cassette" refers to (1) a target DNA sequence (e.g., a polynucleotide capable of functioning to encode TVP) and one or more of the following: (2) a promoter, terminator, and / or enhancer element; (3) a suitable mRNA-stabilizing polyadenylation signal; (4) an intrasequence 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)-(6) is called 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 containing a polynucleotide capable of functioning to encode TVP. In alternative embodiments, there may be two expression cassettes (i.e., a double expression cassette) each containing a polynucleotide capable of functioning to encode TVP. In other embodiments, there may be three expression cassettes (i.e., a triple expression cassette) capable of functioning to encode TVP. In some embodiments, a dual expression cassette may be produced by subcloning a second expression cassette into a vector containing a first expression cassette. In some embodiments, a triple expression cassette may be produced by subcloning a third expression cassette into a vector containing a first expression cassette and a second expression cassette. Methods relating to expression cassettes and cloning techniques are well known in the art and are described herein. See also TVP expression cassettes.

[0073] "Expressed ORF" refers to nucleotides that encode a protein complex, and is defined as nucleotides within an ORF.

[0074] "FECT" refers to a transient plant expression system that uses foxtail mosaic virus with the coating protein gene and triple gene block excluded.

[0075] "Fermented beer" refers to the consumed fermentation medium, i.e., the supernatant of the fermentation medium after bioremoval, in which transformed host cells are seeded and consumed by said transformed host cells (e.g., yeast cells capable of expressing the TVP of the present invention). In some embodiments, fermented beer refers to the solution recovered after fermentation of the transformed host cells. The term "fermentation" broadly refers to the enzymatic and anaerobic or aerobic decomposition of organic matter (e.g., carbon substrates) nutrients by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, and the like) to produce fermentation products (e.g., one or more of the peptides of the present invention). While fermentation typically describes a process that occurs under anaerobic conditions, the term is not intended to be limited to strictly anaerobic conditions as used herein, and the term "fermentation" as used herein may also apply to processes that occur in the presence of enzymes.

[0076] "Fermented solids" refers to the solids (including dissolved solids) that remain in the fermented beer during the yeast-based fermentation process and consist of salts, complex protein sources, vitamins, and additional yeast byproducts with a molecular weight cutoff of approximately 200 kDa to approximately 1 kDa.

[0077] "GFP" stands for green fluorescent protein derived from the jellyfish (Aequorea victoria).

[0078] "Homologie" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a certain position in both of two comparison sequences is occupied by the same base monomer subunit or amino acid monomer subunit, for example, if a certain position in each of two DNA molecules is occupied by adenine, then those molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matched or homologous positions shared by those two sequences divided by the number of comparison positions, multiplied by 100. Therefore, in some embodiments, the term "homologie" refers to sequence similarity between two polypeptide molecules or two nucleic acid molecules. If a certain position in both of two comparison sequences is occupied by the same base monomer subunit or amino acid monomer subunit, for example, if a certain position in each of two DNA molecules is occupied by adenine, then those molecules are homologous at that position. The homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences. For example, if two sequences have 10 positions, and 6 of those positions match or are homologous, then those two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology.

[0079] The term "homology," when used in relation to nucleic acids, refers to the degree of complementarity. Homology can be partial or complete (i.e., identical). "Sequence identity" refers to a measure of the relationship between two or more nucleic acids, given as a percentage of the total comparative length. In calculating identity, nucleotide residues that are identical at the same relative position in each longer sequence are considered.

[0080] "Homologous recombination" refers to the phenomenon in which a segment of DNA is replaced by another segment that has an identical (homologous) or nearly identical region. For example, in some embodiments, "homologous recombination" refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar DNA molecules or between two identical DNA molecules. Briefly, homologous recombination is most widely used by cells to precisely repair harmful breaks (known as double-strand breaks) that occur on both strands of DNA. Although homologous recombination varies considerably between different organisms and cell types, most forms follow the same basic steps: after a double-strand break occurs, the DNA section near the 5' end of the break is detached in a process called excision. In the subsequent strand entry step, the protruding 3' end of the broken DNA molecule "enters" an unbroken, similar, or identical DNA molecule. After strand entry, a series of further events may occur that may follow one of two main pathways: namely, the double-strand break repair pathway or the synthesis-dependent strand annealing pathway. Homologous recombination is conserved across all three biological domains and viruses, suggesting that it is a nearly universal biological mechanism. For example, in some embodiments, homologous recombination may occur using site-directed integration (SSI) sequences, thereby resulting in a strand-swapping crossover event between nucleic acid sequences with substantially similar nucleotide compositions. Such crossover events may occur between sequences contained in the target-directed construct of the present invention (i.e., SSI sequences) and endogenous genomic nucleic acid sequences (e.g., polynucleotides encoding peptide subunits). Furthermore, in some embodiments, multiple site-directed homologous recombinations may occur, resulting in substitution events in which multiple nucleic acid sequences contained within the target-directed construct replace specific sequences present in the endogenous genomic sequence.

[0081] "Identity" refers to the association between two or more polypeptide sequences or two or more polynucleotide sequences, which is determined by comparing these sequences. The term "identity" also refers to the degree of sequence association between polypeptide sequences or polynucleotide sequences, which is sometimes determined by matching the strands of such sequences. "Identity" and "similarity" can be readily calculated by any one of countless methods known to those skilled in the art, including, but not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied This includes the information found in Math., 48:1073 (1988), and the disclosures of these documents are incorporated herein by reference in their entirety. Furthermore, methods for determining identity and similarity are coded into publicly available computer programs.For example, in some embodiments, methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BLASTP, BLASTN, and FASTA (Altschul, S. et al., J. Molec. Biol. 215:403-410(1990). The BLAST X program is publicly available from NCBI and other suppliers (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410(1990)), and the disclosures of such literature are incorporated herein by reference in their entirety.

[0082] "In vivo" refers to natural environments (e.g., animals or cells), and processes or reactions that occur within those natural environments.

[0083] "Inactive" refers to a state in which something is not in use (e.g., dormant and / or non-functional). For example, when used in relation to a gene, or when there is a reference to a gene, the term inactive means that the gene product is no longer actively synthesized from the gene, the gene product is no longer actively translated into a protein, or in other ways the gene is no longer actively performing its normal function. For example, in some embodiments, the term inactive may refer to the absence of a gene being transcribed into RNA, the absence of RNA processing (e.g., pre-mRNA processing, RNA splicing, or other post-transcriptional modifications), impaired non-coding RNA maturation, impaired RNA transport (e.g., transport from the nucleus to the cytoplasm), impaired translation, impaired protein folding, impaired transposition, impaired protein transport, and / or the suppression and / or impairment of any of the molecules, polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors involved in any of the aforementioned processes.

[0084] "Inoperable" refers to a state in which something does not function, is malfunctioning, or is no longer able to function. For example, when used in relation to a gene, or when there is a reference to a gene, the term inoperable means that the gene is no longer able, permanently or transiently, to function as it would normally. For example, in some embodiments, "inoperable" means that a gene is no longer able to synthesize a gene product, or that the gene product is no longer translated into a protein, or in other ways the gene is unable to perform its normal function. For example, in some embodiments, the term inoperable may mean the absence of a gene being transcribed into RNA, the absence of RNA processing (e.g., pre-mRNA processing, RNA splicing, or other post-transcriptional modifications), impaired non-coding RNA maturation, impaired RNA transport (e.g., transport from the nucleus to the cytoplasm), impaired translation, impaired protein folding, impaired transposition, impaired protein transport, and / or the suppression and / or impairment of any of the molecules, polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors involved in any of the aforementioned processes.

[0085] "Insects" includes all organisms of the class "Insecta". The term "juvenile adult" refers to any form of organism prior to the adult stage (e.g., eggs, larvae, and nymphs). As used herein, "insects" refers to any arthropod and nematode (including mites, and all insects known to infest crops, vegetables, and trees), and includes insects considered pests in the fields of forestry, horticulture, and agriculture. Examples of specific crops that may be protected in the manner disclosed herein are soybeans, maize, cotton, alfalfa, and vegetable crops. A list of specific crops and insects is provided herein.

[0086] "Insecticidal activity" means that, upon or after exposure to a compound, agent, or peptide, the insect dies, stops or slows down its activity, stops or slows down its feeding, stops or slows down its growth, becomes confused (e.g., with respect to course determination, foraging, sleep behavior, and / or mating), fails to pupate, develops reproductive disorders, and / or is prevented from producing offspring, and / or is prevented from producing reproductive offspring.

[0087] "Insect intestinal environment" or "intestinal environment" refers to the specific pH and proteinase conditions found in the foregut, midgut, or hindgut of insects or insect larvae.

[0088] The term "insect hemolymphatic environment" refers to specific pH and proteinase conditions found within insects or insect larvae.

[0089] An "integrated expression vector" or "integrated vector" refers to a yeast expression vector that can be inserted into a specific gene locus in the yeast cell genome and stably become part of the yeast genome.

[0090] An "intermediate linker" refers to a short peptide sequence within a protein that separates different parts of the protein, or a short DNA sequence placed in the reading frame of an ORF to separate an upstream DNA sequence from a downstream DNA sequence. For example, in some embodiments, the use of an intermediate linker may allow the independent formation of secondary and tertiary structures in a protein during translation. In some embodiments, the intermediate linker may be resistant or susceptible to cleavage in plant cell environments, insect and / or lepidopteran intestinal environments, as well as insect and lepidopteran hemolymphatic environments.

[0091] "Isolated" means that a thing and / or component has been separated from its natural environment. For example, a toxin isolated from a given genus or species means that the toxin has been separated from its natural environment.

[0092] "kb" refers to a kilobase, or 1000 base pairs. 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 with a length of 1000 nucleotides. A double-stranded DNA molecule with a length of 1 kb contains 2000 nucleotides (i.e., 1000 on each strand). Alternatively, a single-stranded RNA molecule with a length of 1 kb contains 1000 nucleotides.

[0093] "kDa" refers to a kilodalton (a unit equivalent to 1,000 daltons). "Dalton" is a unit of molecular weight (MW).

[0094] "Knock-in," "knock-in," "knocks-in," or "knocking-in" refers to the substitution of an endogenous gene with a foreign or heterologous gene or a portion thereof. For example, in some embodiments, the term "knock-in" refers to introducing a nucleic acid sequence encoding a desired protein into a target gene locus by homologous recombination, thereby causing the expression of the desired protein. In some embodiments, altering a gene sequence by a "knock-in" mutation can create loss-of-function or gain-of-function mutations. The term "knock-in" may refer to the procedure of introducing a foreign polynucleotide sequence or a heterologous polynucleotide sequence or a fragment thereof into a genome (e.g., "a knock-in was performed" or "a heterologous gene was knocked in"), or to the resulting cell and / or organism (e.g., "the cell is knock-in" or "the animal is knock-in").

[0095] "Knockout," "knockout," "knock-out," or "knocking-out" refers to the partial or complete suppression of the expression of a gene product (e.g., mRNA) of a protein encoded by an endogenous DNA sequence in a cell. In some embodiments, "knockout" can be achieved by targeting and deleting an entire gene, or a portion of a gene that codes for a peptide, polypeptide, or protein. As a result, this deletion may cause the gene to become inactive, partially inactive, non-functional, partially non-functional, or otherwise reduce the expression of the gene or its product in any cell in the organism and / or cell in which the gene is normally expressed. The term "knockout" may refer to the procedure of completely or partially inactivating or non-functionalizing an endogenous gene (e.g., "a knockout was performed" or "the endogenous gene was knocked out"), or to the resulting cell and / or organism (e.g., "the cell is knockout" or "the animal is knockout").

[0096] "50% knockdown dose" or "KD 50 " refers to the median dose required to paralyze or halt the activity of 50% of a population (e.g., a population of Musca domestica (common houseflies) and / or Aedes aegypti (mosquitoes)).

[0097] "l" or "linker" refers to a nucleotide that codes for an intervening linker peptide.

[0098] In the appropriate context, "L" refers to an intervening linker peptide, which links a translation-stabilizing protein (STA) with additional polypeptides (e.g., one TVP and / or more TVPs). If there is a reference to an amino acid, "L" can also mean leucine.

[0099] The "LAC4 promoter" or "Lac4 promoter" refers to a DNA segment consisting of a promoter sequence derived from the β-galactosidase gene of K. lactis. The LAC4 promoter is a potent inducible reporter used to guide the expression of transformed exogenous genes in yeast.

[0100] The "LAC4 terminator" or "Lac4 terminator" refers to a DNA segment composed of a transcriptional terminator sequence derived from the β-galactosidase gene of K. lactis.

[0101] "LD 20 " refers to the dose required to kill 20% of the population.

[0102] "LD 50 "50% lethal dose" refers to the dose required to kill 50% of a population.

[0103] The term "lepidopteran intestinal environment" refers to the specific pH and proteinase conditions found within the foregut, midgut, or hindgut of lepidopteran insects or larvae.

[0104] The term "lepidopteran hemolymphatic environment" refers to specific pH and proteinase conditions found within lepidopteran insects or their larvae.

[0105] A “linker,” “LINKER,” “peptide linker,” “L,” or “intermediate linker” refers to a short peptide sequence capable of functioning to link two peptides together. A linker may also refer to a short DNA sequence placed in the reading frame of an ORF to separate an upstream DNA sequence from a downstream DNA sequence. In some embodiments, linkers may be cleavable by insect proteases. In some embodiments, linkers may be capable of independently forming secondary and tertiary structures in a protein during translation. In some embodiments, linkers may be resistant or sensitive to cleavage in plant cell environments, insect and / or lepidopteran intestinal environments, and / or insect and lepidopteran hemolymphatic environments. In some embodiments, linkers may be cleaved by proteases. For example, in some embodiments, the linker may be cleaved by plant proteases (e.g., papain, bromelain, ficin, actinidin, zingipain, and / or cardosin), insect proteases, fungal proteases, vertebrate proteases, invertebrate proteases, bacterial proteases, mammalian proteases, reptile proteases, or avian proteases. In some embodiments, the linker may be cleavable or incleavable. In some embodiments, the linker includes a binary or tertiary region, each region being cleavable by at least two types of proteases: one of these proteases is an insect and / or nematode protease, and the other is a human protease. In some embodiments, the linker may have one of three roles (at least) (to be cleaved in the insect intestinal environment, to be cleaved in plant cells, and to be designed so that unintended cleavage does not occur).

[0106] "Medium" (plural: "media") refers to the nutrient solution used to culture cells in cell culture.

[0107] "MOA" refers to the mechanism of action.

[0108] "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 carried out as follows: The sample of interest is separated on a gel along with a series of molecular weight standards. After electrophoresis of the sample, the gel is subjected to the desired staining treatment, followed by a destaining treatment for approximately 2 to 14 hours. In the next step, the relative migration distance (Rf) of the standards and the protein of interest is determined. The migration distance can be determined using the following formula:

number

[0109] Next, the logarithm of the molecular weight (MW) can be determined based on the values ​​obtained from the standard material band. For example, in some embodiments, the logarithm of the molecular weight of the SDS-modified polypeptide and the relative electrophoretic distance (Rf) of the SDS-modified polypeptide are plotted on a graph. After plotting the graph, the molecular weight of the unknown protein band can be obtained by interpolating the obtained values.

[0110] A "motif" refers to a polynucleotide or polypeptide sequence that is involved in having some biological significance and / or exerting some effect or participating in some biological process.

[0111] A "multi-cloning site" or "MCS" refers to a DNA segment found in a vector that contains multiple restriction enzyme recognition sites into which the target DNA sequence can be inserted.

[0112] A “mutant” means an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein having a modification or difference (e.g., in a nucleotide sequence or amino acid sequence) such modification or difference 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 mutant is compared. In some embodiments, this modification or difference may be one or more nucleotide and / or amino acid substitutions or modifications (e.g., deletions or additions). In some embodiments, one or more amino acid substitutions or modifications may be conserved. In this specification, such conserved amino acid substitutions and / or modifications in a “mutant” do not substantially impair the activity of the mutant in relation to its non-mutant form. For example, in some embodiments, a “mutant” has one or more conserved amino acid substitutions when compared to a peptide having the disclosed and / or claimed sequence (indicated by the sequence number).

[0113] The "N-terminus" refers to the free amine group (i.e., -NH2) located at the beginning or starting point of a polypeptide.

[0114] "NCBI" refers to the National Center for Biotechnology Information in the United States.

[0115] "nm" refers to nanometers.

[0116] "Normalized peptide yield" refers to the peptide yield in the acclimatization medium divided by the corresponding cell density at the time the peptide yield is measured. Peptide yield can be expressed as the mass of the produced peptide per volume (e.g., mg per liter, i.e., mg / L) or as the UV absorbance peak area of ​​the produced peptide in HPLC chromatography (e.g., mAu.sec). Cell density can be expressed as the visible light absorbance of the culture at a wavelength of 600 nm (OD600).

[0117] "OD" refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring the growth of a cell population over time, OD600 is preferred over UV spectroscopy. This is because the harm that would occur under excessive UV light does not occur to cells at a wavelength of 600 nm.

[0118] "OD660nm" or "OD 660nm This refers to the optical density at 660 nanometers (nm).

[0119] "Single-letter abbreviation" refers to the use of single-letter abbreviations to represent peptide sequences and distinguish the various amino acids in the primary structure of proteins: 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.

[0120] "Functional" refers to the ability to be used, the ability to do something, and / or the ability to achieve some function or result. For example, in some embodiments, "functional" refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence, or gene, to code for peptides, polypeptides, and / or proteins. For example, in some embodiments, a polynucleotide may be functional to code for a protein, meaning that the polynucleotide contains information that gives it the ability to create a protein (e.g., by transcription of mRNA and subsequent translation into a protein).

[0121] "Functionally linked" means that the elements described in this way are arranged in a relationship that allows them to function in the intended manner. For example, in some embodiments, functionally linked may refer to two or more DNA sequences, peptide sequences, or polypeptide sequences. For example, in some embodiments, functionally linked may mean that two adjacent DNA sequences are arranged relative to each other so that the transcriptional activation of one adjacent DNA sequence can act on the other adjacent DNA sequence. In other embodiments, the term "functionally linked" may refer to peptide molecules and / or polypeptide molecules, for example, functionally linked may mean that two or more peptide molecules and / or polypeptide molecules are linked in such a way that they give a single polypeptide chain, or so that one peptide has some effect on the other peptide. In yet another embodiment, functionally linked may mean that two adjacent DNA sequences are arranged relative to each other so that the transcriptional activation of one adjacent DNA sequence can act on the other adjacent DNA sequence. In other embodiments, functionally linked may refer to peptide molecules and / or polypeptide molecules, where two or more peptide molecules and / or polypeptide molecules are linked in such a manner that they give a single polypeptide chain, or so that one peptide has some effect on the other peptide.

[0122] An "ORF" or "open reading frame" refers to an RNA or DNA sequence of a length that encodes one or more polypeptide sequences, such sequences are located between a translation start signal (e.g., AUG or ATG, respectively) and one or more known stop codons. In other words, an ORF describes a baseline frame in which a ribosome translates an RNA code as long as it can continue reading (i.e., adding amino acids to a nascent protein) because it has not yet reached a stop codon. Thus, an "open reading frame" or "ORF" refers to the amino acid sequence encoded between the translation start codon and the stop codon of a coding sequence. In this specification, the terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence, which defines the start and termination of protein synthesis (mRNA translation), respectively.

[0123] In some embodiments, an ORF is a sequence of codons beginning with a start codon (typically ATG for DNA and AUG for RNA) and ending with a stop codon (typically UAA, UAG, or UGA). In other embodiments, an ORF may be an RNA or DNA sequence of a length between a translation initiation signal (e.g., AUG or ATG) and one or more known stop codons, where the RNA or DNA sequence of this length codes for one or more polypeptide sequences. In some other embodiments, an ORF may be a protein-coding DNA sequence beginning with an ATG start codon and ending with a TGA, TAA, or TAG stop codon. An ORF may also refer to the translated protein encoded by such DNA. Generally, those skilled in the art distinguish "open reading frame" and "ORF" from the term "coding sequence" based on the fact that the broadest definition of "open reading frame" is simply a sequence of codons that does not contain a stop codon. Therefore, while ORFs may contain introns, coding sequences are distinguished by referring to nucleotides (e.g., chained exons) that can be classified as codons that are actually translated into amino acids by the ribosome translation mechanism (i.e., coding sequences do not contain introns); however, as used herein, the terms “coding sequence,” “CDS,” “open reading frame,” and “ORF” are used interchangeably.

[0124] "Recombination-removed" or "recombination-removed" refers to the removal of two site-specific recombination sites (e.g., the 5' and 3' nucleotide sequences of the target gene, homologous to the homology arms of the target vector) and adjacent genes and / or polynucleotide sequences (e.g., endogenous genes) during homologous recombination in vivo. See "knockout".

[0125] "Pests" include, but are not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and similar organisms.

[0126] "An effective amount as a pesticide" refers to the amount of pesticide that can kill at least one pest, or that can significantly reduce the growth, feeding, or normal physiological development of the pest. This amount will vary depending on several factors, such as the specific target pest being controlled, the specific environment, location, plant, crop, or agricultural land being treated, environmental conditions, and the method, proportion, concentration, stability, and amount of application of the effective polypeptide composition as a pesticide. Formulations may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of pest spread.

[0127] "Medically acceptable salt" is synonymous with "agriculturally acceptable salt," and as used herein, refers to a compound modified by preparing its acid salt or base salt.

[0128] "Plant" means the entire plant, its plant tissues, plant organs (e.g., leaves, stems, roots), seeds, plant cells, bulbils, embryos, and offspring. Plant cells may be differentiated or undifferentiated (e.g., callus, suspension cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0129] "Plant transgenic protein" refers to a heterologous protein that is expressed in a plant after the DNA or RNA encoding that heterologous protein has been delivered to one or more plant cells.

[0130] A plasmid is a DNA segment that acts as a carrier for a target gene (e.g., tvp) and, when transformed or transfected into an organism, can replicate and express its own DNA sequence independently of the host organism. A plasmid is a type of vector and can be a "cloning vector" (i.e., a simple plasmid used for cloning DNA fragments and / or selecting plasmid-bearing host populations via some selection criterion) or an "expression plasmid" (i.e., a plasmid used for the mass production of polynucleotides and / or polypeptides).

[0131] "Polynucleotide" refers to a polymeric form of nucleotide (e.g., ribonucleotide, deoxyribonucleotide, or analogue thereof) of any length, such nucleotides being, for example, a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term "polynucleotide" includes double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA. The term also includes modified and unmodified forms of polynucleotides (modifications to and from polynucleotides may include, for example, methylation, phosphorylation, and / or capping). In some embodiments, a polynucleotide may be one of the following: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the aforementioned.

[0132] In yet another embodiment, the polynucleotide may refer to a polymeric form of nucleotide capable of encoding the open reading frame of a gene.

[0133] In some embodiments, polynucleotides may refer to cDNA.

[0134] In some embodiments, polynucleotides can have any three-dimensional structure and can exhibit any known or unknown functions. The structure of a polynucleotide can also be referred to by its 5' or 3' end or 5' or 3' end, which indicates 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' carbon atoms. However, different nucleotide bonds (including methylene, phosphoramide, etc.) may also be used. This means that each 5' and 3' carbon can be exposed at either end of the polynucleotide, and these ends may be called the 5' end or 3' end or 5' or 3' end. The 5' and 3' ends may also be called the phosphoryl (PO4) end and hydroxyl (OH) end, respectively, due to the chemical groups attached to them. The term polynucleotide also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of preparing or using polynucleotides encompasses both the double-stranded form and each of the two complementary single-stranded forms known or expected to form the double-stranded form.

[0135] In some embodiments, polynucleotides may include modified nucleotides (such as methylated nucleotides) and nucleotide analogs (including nucleotides with non-native bases and nucleotides with modified native bases (such as azapurines or deazapurines)). Modifications to the nucleotide structure, if present, may be conferred before or after the construction of the polynucleotide.

[0136] In some embodiments, polynucleotides may be further modified after polymerization. This modification is carried out by methods such as attaching labeling elements. Furthermore, the sequence of nucleotides in a polynucleotide may be separated by non-nucleotide elements. One or more ends of a polynucleotide may be protected or otherwise modified to prevent those ends from interacting with other polynucleotides in a particular manner (e.g., forming covalent bonds).

[0137] In some embodiments, polynucleotides may consist of a specific sequence of four nucleotide bases (adenine (A), cytosine (C), guanine (G), and thymine (T)). Uracil (U) may also be present, for example, as a natural thymine substitute when the polynucleotide is RNA. Uracil may also be used in DNA. Therefore, the term “sequence” refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.

[0138] The term "RNA molecule" or "ribonucleic acid molecule" refers to a polynucleotide that has a ribose sugar rather than a deoxyribose sugar, and typically has uracil rather than thymine as one of its pyrimidine bases. The RNA molecules of this invention are usually single-stranded, but may also be double-stranded. In the context of RNA molecules derived from RNA samples, RNA molecules may include single-stranded molecules transcribed from DNA in the cell nucleus, mitochondria, or chloroplasts, which have a linear sequence of nucleotide bases complementary to the DNA strand from which they were transcribed.

[0139] In some embodiments, the polynucleotide may further include one or more heterogeneous regulatory elements. For example, in some embodiments, the regulatory elements may be one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA transport elements, internal ribosome entry sites (IRESs), poly-U sequences, or combinations thereof.

[0140] "Post-transcriptional regulatory elements" are DNA segments and / or mechanisms that influence mRNA after transcription. Mechanisms of post-transcriptional mechanisms include splicing events, capping, splicing, and poly(A) tail addition, as well as other mechanisms known to those skilled in the art.

[0141] A "promoter" refers to the DNA region where RNA polymerase binds and gene transcription begins.

[0142] In this document, "protein" has the same meaning as "peptide" and / or "polypeptide."

[0143] A "ratio" refers to a quantitative relationship between two quantities, indicating the number of times one value contains the other value, or the number of times one value is included in the other value.

[0144] A "reading frame" refers to one of the six possible reading frames (three for each direction) of a double-stranded DNA molecule. The reading frame used determines which codons are used to code the amino acids within the coding sequence of the DNA molecule. In some embodiments, a reading frame is a method of dividing the sequence of nucleotides in a polynucleotide and / or nucleic acid (e.g., DNA or RNA) into a series of consecutive non-overlapping triplets.

[0145] "Recombinant DNA" or "rDNA" refers to DNA composed of two or more different DNA segments.

[0146] A "recombinant vector" refers to a DNA plasmid vector into which foreign DNA has been inserted.

[0147] A “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 may be found at the transcriptional and post-transcriptional levels. Regulatory elements may be cis-regulatory elements (CREs) or trans-regulatory elements (TREs). In some embodiments, regulatory elements may be one or more promoters, enhancers, silencers, operators, splicing signals, polyadenylation signals, termination signals, RNA transport elements, internal ribosome entry sites (IRESs), poly-U sequences, and / or other elements that influence gene expression (e.g., in a tissue-specific or time-dependent manner) to increase or decrease expression and / or cause constitutive expression.

[0148] A "restriction enzyme" or "restriction endonuclease" refers to an enzyme that cleaves DNA at a specific restriction enzyme recognition site. For example, restriction enzymes can linearize a plasmid and ligate the target DNA by cleaving it at the EcoRI, SacII, or BstXI restriction enzyme recognition sites.

[0149] A "restriction enzyme recognition site" refers to a location on DNA that contains a sequence of 4 to 8 nucleotides and is recognized by a specific restriction enzyme.

[0150] A "selection gene" refers to a gene that gives a genetically modified organism an advantage in reproducing under selective pressure.

[0151] A "serovar" or "serotype" refers to a group of closely related microorganisms distinguished by a characteristic set of antigens. In some embodiments, a serovar is an antigenically and serologically distinct variant of a microorganism.

[0152] "sp." refers to one type.

[0153] "ssp." or "subsp." refers to a subspecies.

[0154] "Subcloning" or "subcloned" refers to the process of transferring DNA from one vector to another (usually a favorable vector). For example, a polynucleotide encoding the mutant TVP can be subcloned into the pKlac1 plasmid after selecting yeast colonies transformed with the pKLAC1 plasmid.

[0155] "SSI" is a context-dependent acronym. In some contexts, SSI can refer to "site-directed integration," which is used to describe the ability of a sequence to undergo homologous recombination in vivo. In other contexts, SSI can refer to "indoor surface spraying," a technique that applies a variable volume of insecticide, with a sprayable volume, to indoor surfaces (walls, windows, floors, and ceilings, etc.) where the vector rests. The term "site-directed integration" refers to the process of guiding a transgene to a target site in the host organism's genome; therefore, SSI enables the integration of the target gene into a pre-selected host organism genome location.

[0156] "STA," or "translation-stabilizing protein," or "stabilizing domain," or "stabilizing protein" (as used interchangeably herein) means a peptide or protein having sufficient tertiary structure to accumulate in cells without being targeted by cellular processes of proteolysis. The amino acid length of this protein may be 5 to 50. The translation-stabilizing protein is encoded by a protein DNA sequence functionally ligated to a sequence encoding an insecticidal protein or TVP in the ORF. The functionally ligated STA may be located either upstream or downstream of the TVP and may have any intervening sequence between these two sequences (STA and TVP), provided that this intervening sequence does not cause a frameshift of either DNA sequence. The translation-stabilizing protein may also have activity that increases the delivery of TVP across the intestinal wall to the insect hemolymph.

[0157] "sta" refers to a nucleotide that codes for translation stabilization proteins.

[0158] A "structural motif" refers to the three-dimensional arrangement of a peptide and / or polypeptide, and / or the arrangement of functionally linked polypeptide segments. For example, a polypeptide having the ERSP motif, STA motif, LINKER motif, and TVP polypeptide motif has the overall "structural motif" of ERSP-STA-L-TVP. See also "TVP construct".

[0159] "Ta1b," "U1-agatoxin-Ta1b," "Ta1bWT," or "wild-type U1-agatoxin-Ta1b" refers to polypeptides isolated from the genus Eratigena agrestis. An example of U1-agatoxin-Ta1b is the polypeptide with the amino acid sequence of Sequence ID No. 1 (NCBI acceptance number O46167.1).

[0160] "Ta1b variant polynucleotide" or "U1-agatoxin-Ta1b variant polynucleotide" refers to a polynucleotide or group of polynucleotides capable of functioning to express and / or encode an insecticidal protein containing one or more TVPs. The term "U1-agatoxin-Ta1b variant polynucleotide" is used to describe a U1-agatoxin-Ta1b variant polynucleotide sequence contained in a TVP expression ORF, to describe its inclusion in a vector, and / or to describe a polynucleotide encoding an insecticidal protein, and is therefore referred to as "tvp" and / or "Tvp".

[0161] "Toxin" refers to venomous fluids and / or poisonous substances, and in particular to proteins or complex proteins produced by certain animals, higher plants, and pathogenic bacteria. Generally, the term "toxin" is used for natural products (e.g., molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc.), while the term "toxic substance" is used for artificial products and / or concoctions (e.g., artificial chemical pesticides). However, as used herein, the terms "toxin" and "toxic substance" are used synonymously.

[0162] Both “transfect” and “transformation” refer to the process of introducing foreign and / or heterologous DNA or RNA (e.g., a vector containing polynucleotides encoding TVP) into a host organism (e.g., a prokaryote or a eukaryote). Generally, those skilled in the art may use the term “transformation” to describe the process of introducing foreign and / or heterologous DNA or RNA into bacterial cells, and the term “transfect” to describe the process of introducing foreign and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms “transformation” and “transfect” are used synonymously regardless of whether the target organism to which the foreign and / or heterologous DNA or RNA described by the process is a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plant, or animal).

[0163] "Transgene" refers to a protein-coding heterologous DNA sequence that is transformed into a plant.

[0164] A "transgenic host cell" refers to a cell in which the genes have been transformed and that transgenic state has been selected through additional selection genes.

[0165] A "transgenic plant" is a plant in which a single cell has been transformed with foreign DNA, and as a result, every cell in the plant contains that transgene.

[0166] A "transient expression system" refers to an Agrobacterium tumefaciens-based system that delivers DNA encoding a disarming plant virus to plant cells where it is expressed. This plant virus is engineered to express the target protein at high concentrations (up to 40% in the case of TSP).

[0167] A "triple expression cassette" refers to three TVP expression cassettes contained within the same vector.

[0168] "TRBO" refers to a transient plant expression system that uses tobacco mosaic virus from which the virus coating protein gene has been removed.

[0169] "TSP" or "Total Soluble Protein" refers to the total amount of protein that can be extracted from a plant tissue sample, solubilized, and included in the extraction buffer.

[0170] "TVP" or "U1-agatoxin-Ta1b variant polypeptide (TVP)" or "Ta1b variant polypeptide (TVP)" refers to a variant or variant in which the wild-type U1-agatoxin-Ta1b polypeptide sequence and / or the polynucleotide sequence encoding the wild-type U1-agatoxin-Ta1b polypeptide has been modified to yield a non-natural polypeptide sequence and / or polynucleotide sequence. An example of a wild-type U1-agatoxin-Ta1b polypeptide sequence is provided herein and has the amino acid sequence of SEQ ID NO: 1. An example of a wild-type U1-agatoxin-Ta1b precursor polypeptide sequence is provided herein and has the amino acid sequence of SEQ ID NO: 48 (NCBI acceptance number O46167.1), which contains a signal sequence of the form "MKLQLMICLVLLPCFFC" (SEQ ID NO: 59). In some embodiments, a TVP may have an amino acid sequence of any of the amino acid sequences listed in Table 1. Thus, the term "TVP" refers to a peptide having one or more mutations compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, TVP is given by equation (I): EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7 Equation (I) It may have the following amino acid sequence:

[0171] In the formula, the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0172] In some embodiments, TVP or a pharmaceutically acceptable salt thereof is Formula (II): EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG Formula (II) It may have the following amino acid sequence:

[0173] In the formula, the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A.

[0174] "TVP ORF schematic diagram" refers to the composition of one or more TVP ORFs described in schematic or formulaic form. For example, a "TVP ORF schematic diagram" can be described using acronyms or abbreviations for DNA segments contained within an expression ORF. Thus, in one example, in a "TVP ORF schematic diagram", the polynucleotide segment encoding ERSP, the polynucleotide segment encoding LINKER, the polynucleotide segment encoding STA, and the polynucleotide segment encoding TVP can be described by diagramming those 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 "tvp" (i.e., the polynucleotide sequence encoding TVP), respectively. An example of a TVP ORF schematic diagram is " i -tvp j ) N " or " j -linker i ) N -sta", and / or any combination of those DNA segments.

[0175] "TVP polynucleotide" refers to a polynucleotide or group of polynucleotides capable of functioning to express and / or encode TVP or a TVP-insecticidal protein.

[0176] "TVP-insecticide protein" means any protein, peptide, polypeptide, amino acid sequence, composition, or arrangement comprising (1) at least one TVP or two or more TVPs, and (2) an additional non-toxic peptide, polypeptide, or protein, wherein the additional non-toxic peptide, polypeptide, or protein is capable of doing one or more of the following, for example, in some embodiments: causing increased insect mortality and / or growth inhibition when insects are exposed to the TVP-insecticide protein compared to TVP alone; increasing the expression of the TVP-insecticide protein (e.g., in host cells or expression systems); and / or affecting the post-translational processing of the TVP-insecticide protein. In some embodiments, the TVP-insecticide protein may be a polymer comprising two or more TVPs. In some embodiments, the TVP-insecticide protein may be a polymer comprising two or more TVPs, where the TVPs are functionally linked via linker peptides (e.g., cleavable and / or non-cleavable linkers). In some embodiments, the TVP-insecticide protein may refer to one or more TVPs functionally linked to one or more proteins, the one or more of which are stabilizing domains (STAs), endoplasmic reticulum signaling proteins (ERSPs), insect-cleavable or insect-incapable linkers (L), and / or any other combination thereof. In some embodiments, the TVP-insecticide protein may be a non-natural protein comprising (1) a wild-type Ta1b protein and (2) an additional non-toxic peptide, polypeptide, or protein (e.g., ERSP, linker, STA, UBI, or histidine tag or similar marker).

[0177] The term "TVP construct" refers to the three-dimensional arrangement / orientation of peptides, polypeptides, and / or motifs (e.g., TVP-insecticide proteins) of functionally linked polypeptide segments. For example, a TVP expression ORF may contain one or more of the following elements or motifs: TVP, endoplasmic reticulum signal peptide (ERSP), linker peptide (L), translation stabilizing protein (STA), or any combination thereof. Furthermore, as used herein, the term "TVP construct" is also used to describe the designation and / or orientation of structural motifs. In other words, a TVP construct describes the arrangement and orientation of elements or motifs contained within a given TVP expression ORF. For example, in some embodiments, the TVP construct describes the orientation of one of the following TVP-insecticide proteins, but is not limited to: ERSP-TVP, ERSP-(TVP) N ERSP-TVP-L, ERSP-(TVP) N -L, ERSP-(TVP-L) N ERSP-L-TVP, ERSP-L-(TVP) N ERSP-(L-TVP) N , ERSP-STA-TVP, ERSP-STA-(TVP) N ERSP-TVP-STA, ERSP-(TVP) N -STA, ERSP-(STA-TVP) N ERSP-(TVP-STA) N , ERSP-L-TVP-STA, ERSP-L-STA-TVP, ERSP-L-(TVP-STA) N ERSP-L-(STA-TVP) N ERSP-L-(TVP) N -STA, ERSP-(L-TVP) N -STA, ERSP-(L-STA-TVP) N ERSP-(L-TVP-STA) N ERSP-(L-STA) N -TVP, ERSP-(L-TVP) N-STA, ERSP-STA-L-TVP, ERSP-STA-TVP-L, ERSP-STA-L-(TVP) N ERSP-(STA-L) N -TVP, ERSP-STA-(L-TVP) N ERSP-(STA-L-TVP) N ERSP-STA-(TVP) N -L, ERSP-STA-(TVP-L) N ERSP-(STA-TVP) N -L, ERSP-(STA-TVP-L) N , ERSP-TVP-L-STA, ERSP-TVP-STA-L, ERSP-(TVP) N -STA-L, ERSP-(TVP-L) N -STA, ERSP-(TVP-STA) N -L, ERSP-(TVP-L-STA) N , or ERSP-(TVP-STA-L) N (In the formula, N is an integer between 1 and 200). See also "Structural Motifs".

[0178] The term "var." refers to a variety or variant. The term "var." is used to indicate a taxonomic category lower than the species level and / or subspecies (if any). In some embodiments, the term "var." refers to a member that exhibits only minor characteristic differences from others of the same subspecies or species, but in which such characteristics are persistent or heritable.

[0179] A “variant,” “variant sequence,” or “variant peptide” refers to an amino acid sequence having one or more conserved amino acid substitutions or modifications. Conservative amino acid substitutions in a “variant” do not substantially impair the activity of the variant in relation to its non-variant form. For example, in some embodiments, a “variant” has one or more conserved amino acid substitutions compared to a peptide having the disclosed and / or claimed sequence (indicated by the SEQ ID NO).

[0180] A "vector" refers to a DNA segment that accepts a foreign target gene (e.g., tvp). The target gene is also known as an "insertion fragment" or "transgene."

[0181] "Vitrification" refers to the process of converting a material into a glassy amorphous material. Glassy amorphous solids cannot possess any crystalline structure. Solidification of glassy solids occurs at the glass transition temperature (Tg).

[0182] "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 seen and / or observed in its naturally occurring state or conditions.

[0183] "Yeast expression vector," "expression vector," or "vector" means a plasmid that can introduce a heterologous gene and / or expression cassette into yeast cells to induce transcription and translation.

[0184] "Yield" refers to the production of peptides, and an increase in yield may mean an increase in production volume, an increase in production rate, as well as an increase in average or median yield and an increase in the frequency of yield improvements. When the term "yield" is used in relation to the growth and / or production of plant crops, as in the usage "plant yield," it refers to the quality and / or quantity of biomass produced by the plant.

[0185] Throughout this specification, unless otherwise specifically stated or the context requires a different interpretation, references to a single step, composition, group of steps, or group of compositions shall encompass one and more (i.e., one or more) such steps, compositions, group of steps, or group of compositions.

[0186] This disclosure is carried out without excessive experimentation using the standard methods of molecular biology, microbiology, virology, recombinant DNA technology, solid-phase nucleic acid synthesis and liquid-phase nucleic acid synthesis, peptide synthesis in solution, solid-phase peptide synthesis, immunology, cell culture, and formulation, unless otherwise specified. Such procedures are described in, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989) (Vols I, II, and III in their entirety), DNA Cloning: A Practical Approach, Vols. I and II (DNGlover, ed., 1985), IRL Press, Oxford (the entire text), Oligonucleotide Synthesis: A Practical Approach (MJ Gait, ed., 1984) IRL Press, Oxford (the entire text, and in particular the articles by Gait (pp. 1-22), Atkinson et al (pp. 35-81), Sproat et al (pp. 83-115), and Wu et al (pp. 135-151) included in the said literature, 4. Nucleic Acid Hybridization: A Practical Approach (BDHames & SJ Higgins, eds., 1985) IRL Press, Oxford (entire text), Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford (entire text), Perbal, B., A Practical Guide to Molecular Cloning (1984), Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) (entire 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.Land 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,3.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 edn.,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)、ならびにAnimal Cell Culture:Practical Approach,Third Edition(John R.W.Masters,ed.These references are listed in (2000), and each of them is incorporated herein by reference in its entirety.

[0187] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations thereof (such as “comprises” or “comprising”) should be understood to imply that it includes the steps or elements or integers or groups of steps or elements or groups of integers described, but does not exclude other steps or elements or integers or groups of elements or groups of integers.

[0188] All patent applications, patents, and printed publications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference in whole. Furthermore, all patent applications, patents, and printed publications cited herein are incorporated by reference in whole, except that any definitions, subject matter exclusions, or disavows are excluded, and also excluded if the incorporated material is inconsistent with the disclosures shown herein, in which case the words of this disclosure shall prevail.

[0189] Wild-type U1-agatoxin and TVP

[0190] *Eratigena agrestis* (formerly *Tegenaria agrestis*) is a venomous spider, or funnel-web spider, belonging to the family Agelenidae. See Ingale A, Antigenic epitopes prediction and MHC binder of a paralytic insecticidal toxin (ITX-1) of *Tegenaria agrestis* (hobo spider). 4 August 2010 Volume 2010:2 pp 97-103. The venom of *Eratigena agrestis* is associated with the possession of 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 See One.2013;8(5):e63865.

[0191] The bitterling spider, along with several other spiders in the family Agelenidae, produces a venom containing agatoxin (which exhibits insecticidal activity). Agatoxin is a chemically diverse group of toxins that can induce various insecticidal effects depending on the target species. For example, agatoxin causes delayed spastic paralysis in coleoptera, lepidoptera, and diptera, increases neuronal firing rate in the central nervous system (CNS) of the housefly (Musca domestica), and is lethal to other insects (e.g., blowflies (Lucilia cuprina)). Therefore, agatoxin is involved in targeting the CNS. 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 See Biochem. Physiol. 38:19-31 (1998).

[0192] Agatoxin includes two forms, U1-agatoxin-Ta1a and U1-agatoxin-Ta1b, both of which are members of the Helix Arthropod Neuropeptide (HAND) toxin family. In addition to spiders, these toxins can also be found in centipede venom. Agatoxin is an evolutionary derivative of the ancient molting animal hormone family, namely the ion transport peptide / crustacean blood glucose-raising hormone (ITP / CHH) family. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators. Structure 23:1283-1292, and Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19-31 (1998).

[0193] The complete amino acid sequence of the U1-agatoxin-Ta1b toxin from the Sachitan spider is "MKLQLMICLVLLPCFFCEPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQKG" (SEQ ID NO: 48). This amino acid sequence contains a signal peptide at amino acid positions 1-17 and the mature toxin at positions 18-68 (see above). This protein contains four tightly packed α-helices, lacks β-strands, and the molecular weight of the mature toxin is 5700.39 daltons (Da) (see above).

[0194] As an example of a mature wild-type U1-agatoxin-Ta1b polypeptide derived from Eratigena agrestis, one having the amino acid sequence "EPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQKG" (Sequence ID 1) is provided.

[0195] In mature wild-type U1-agatoxin-Ta1b toxin, the C-terminal glycine is removed during protein processing, resulting in the following amino acid sequence: EPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQK (Sequence ID 60). Subsequently, a post-translational event occurs, resulting in the production of a mature wild-type U1-agatoxin-Ta1b toxin with an amidated C-terminus.

[0196] U1-agatoxin-Ta1b variant polypeptides (TVPs) are mutants or variants that differ in some way from wild-type U1-agatoxin-Ta1b (SEQ ID NO: 1), for example, in some embodiments, this difference may be an amino acid substitution, deletion, or addition, or a modification to the wild-type U1-agatoxin-Ta1b coding polynucleotide resulting in an amino acid substitution, deletion, or addition. As a result of this difference, a non-natural polypeptide and / or the polynucleotide sequence encoding it is obtained that exhibits enhanced insecticidal activity against one or more insect species compared to wild-type U1-agatoxin-Ta1b.

[0197] In some embodiments, TVP may have the amino acid sequences of SEQ ID NOs: 2-15, 49-53, or 77-110 (shown in Table 1).

[0198] [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

[0199] In some embodiments, the polynucleotide sequence can be functional to encode a TVP having the amino acid sequence of SEQ ID NO: 2-15, SEQ ID NO: 49-53, or SEQ ID NO: 77-110 and can be functional to encode a TVP. For example, in some embodiments, the polynucleotide shown in Table 2 is functional to encode a TVP.

[0200]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

[0201] In some embodiments, the TVP comprises one or more mutations as compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. For example, in some embodiments, the TVP may have a first mutation, a second mutation, or a third mutation as compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1.

[0202] In some embodiments, the TVP may have a first mutation as compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and the first mutation is an amino acid substitution of R9Q, K18A, R38A, A8N, A8S, R9N, T11P, or T43A.

[0203] In some embodiments, the TVP may have a first mutation and a second mutation (e.g., R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, or T43AΔG) as compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, the first mutation is an amino acid substitution of R9Q, K18A, R38A, A8N, A8S, R9N, or T11P, and the second mutation is a deletion of the C-terminal glycine.

[0204] In some embodiments, the TVP may have a first mutation and a second mutation (e.g., R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, or T11PT43A) as compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, the first mutation is an amino acid substitution of R9Q, K18A, R38A, A8N, A8S, R9N, or T11P, and the second mutation is a T43A amino acid substitution that results in a non-glycosylated TVP.

[0205] In some embodiments, TVP may have a first, second, and third mutation (e.g., R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, or T11PT43AΔG) compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where the first mutation is an amino acid substitution of R9Q, K18A, R38A, A8N, A8S, R9N, or T11P, the second mutation is a T43A amino acid substitution resulting in a non-glycosylated TVP, and the third mutation is a deletion of C-terminal glycine.

[0206] In some preferred embodiments, TVP may be TVP-R9Q / T43A (SEQ ID NO: 51).

[0207] In various embodiments, polynucleotides encoding TVP may be used for the transformation of plant cells, yeast cells, or bacterial cells. In some embodiments, insecticidal TVP transgenic proteins may be formulated into compositions that can be sprayed or applied in any other manner known to those skilled in the art to the surface of a plant or a part thereof. Accordingly, DNA constructs capable of functioning to encode one or more TVPs under suitable conditions in a host cell (e.g., a plant cell) are provided herein. A method for controlling insect pest infection of plant cells by parasitic insects comprises administering or introducing the polynucleotides encoding TVP described herein into a plant, plant tissue, or plant cell by a recombinant technique, and growing the recombinantly modified plant, plant tissue, or plant cell in an area exposed to the pest. Alternatively, TVP may be formulated into a sprayable composition comprising TVP and a pharmaceutical additive and applied directly to susceptible plants by direct application, resulting in TVP causing adverse effects upon oral ingestion by infectious insects.

[0208] In some embodiments, TVP may have one of the amino acid sequences of SEQ ID NOs: 2-15, 49-53, and 77-110. In some embodiments, the TVP may include an amino acid sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with the amino acid sequences of SEQ ID NOs. 2-15, SEQ ID NOs. 49-53, or SEQ ID NOs. 77-110.

[0209] In some versions, TVP may contain amino acid sequences that are at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, 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 sequences shown in SEQ ID NOs. 2-15, SEQ ID NOs. 49-53, or SEQ ID NOs. 77-110.

[0210] 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, and less than 50% identical. It may contain 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 amino acid sequences.

[0211] In some embodiments, the TVP may be encoded by a polynucleotide. For example, in some embodiments, the TVP encoded by a polynucleotide or its complementary nucleotide sequence may contain an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the TVP may be the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. Compared to each other, each contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0212] In some embodiments, the polynucleotide encoding TVP may include a polynucleotide in which TVP has one amino acid substitution in X1, X2, X3, X4, or X5.

[0213] In some embodiments, the polynucleotide encoding TVP may comprise a polynucleotide in which TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is glycine.

[0214] In some embodiments, the polynucleotide encoding TVP may consist of a polynucleotide in which TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is absent.

[0215] In some embodiments, the polynucleotide encoding TVP may consist of a polynucleotide in which TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X6 and X7 are absent.

[0216] In some embodiments, the polynucleotide encoding the TVP may comprise a polynucleotide, and the TVP comprises an amino sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0217] In some embodiments, the polynucleotide encoding TVP may include a polynucleotide sequence having the sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150, or a complementary nucleotide sequence.

[0218] In some embodiments, the polynucleotide encoding TVP may consist of a polynucleotide, and if Z1 is T or S, TVP is glycosylated.

[0219] In some embodiments, the polynucleotide encoding TVP may encode a TVP having an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the amino acid sequences shown in SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0220] In some embodiments, the vector may contain a polynucleotide that is capable of functioning to encode TVP.

[0221] In some embodiments, the vector may contain a polynucleotide that is capable of functioning to encode a TVP having an amino acid sequence with 90% similarity to the sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0222] In some embodiments, the vector may contain a polynucleotide having the nucleotide sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150 or its complementary nucleotide sequence.

[0223] Examples of TVP

[0224] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, and at least 96% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7. The TVP may be a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 100% 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, and the polypeptide may be a TVP or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 100% identical compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. It contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0225] 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, and less The TVP or a pharmaceutically acceptable salt thereof may contain an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, and X1 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.

[0226] 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, and at least 98% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7. 1. A TVP or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 X1 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is glycine.

[0227] 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, and at least 98% identical. The TVP may be a pharmaceutically acceptable salt thereof containing an 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 amino acid sequence, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X X1 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; TVP has one amino acid substitution in X1, X2, X3, X4, or X5; and X7 is absent.

[0228] 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, and at least 98% identical. The TVP or a pharmaceutically acceptable salt thereof may contain an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is X1 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X6 and X7 are absent.

[0229] 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, and at least less. The TVP or a pharmaceutically acceptable salt thereof may contain an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, X3 is A, G, N, L, D, V, M, I, C, E, T, or S, X4 is T or P, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP contains an amino sequence shown in any one of sequence numbers 2-15, 49-53, or 77-110.

[0230] 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, and at least 99% identical. The TVP may contain an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S. Yes, 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 or a complementary nucleotide sequence shown in any one of the following sequence numbers: SEQ ID NOs. 17-30, SEQ ID NOs. 54-58, or SEQ ID NOs. 117-150.

[0231] 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, and at least 98% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7. 1. A TVP containing an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, X3 is D, V, M, I, C, E, T, or S, X4 is T or P, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP further comprises two or more homopolymers or heteropolymers of TVP, where the amino acid sequences of each TVP are the same or different.

[0232] 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, and at least 99% identical. The TVP may contain an amino acid sequence that is % 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP is a fusion protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, the amino acid sequences of each TVP may be the same or different.

[0233] 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, and at least 70% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7. 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 The TVP may contain an amino acid sequence that is 100% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, and X3 is T or P. X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP is a fusion protein comprising two or more TVPs separated by a cleavable or incleavable linker, the amino acid sequence of each TVP may be the same or different, and the linker is cleavable in the intestine or hemolymph of insects.

[0234] In some embodiments, the linker has the amino acid sequence shown in any one of SEQ ID NOs: 61-70.

[0235] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, and at least 97% identical. The TVP or a pharmaceutically acceptable salt thereof may contain an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and X1 is TVP is glycosylated if Z1 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.

[0236] 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, The TVP may contain amino acid sequences that are 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.

[0237] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, and at least 9% identical amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG. The TVP may be a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 0% 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A.

[0238] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, and at least 91% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG. TVP or a pharmaceutically acceptable salt thereof containing an amino acid sequence that is 100% 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and if Z1 is T, the TVP is glycosylated.

[0239] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, and at least The TVP or a pharmaceutically acceptable salt thereof may contain an amino acid sequence that is 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, wherein the polypeptide contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, Z1 is T or A, X1 is Q, and Z1 is A.

[0240] In some embodiments, the insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP) is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, and at least 87% identical to the amino acid sequence shown in any one of SEQ ID NOs: 2, SEQ ID NOs: 49, or SEQ ID NOs: 51. TVP or a pharmaceutically acceptable salt thereof may contain amino acid sequences that are 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.

[0241] TVP - Insecticide Protein

[0242] A TVP-insecticide protein is any protein, peptide, polypeptide, amino acid sequence, composition, or arrangement comprising (1) at least one TVP or two or more TVPs, and (2) an additional non-toxic peptide, polypeptide, or protein, the additional non-toxic peptide, polypeptide, or protein having the ability, for example in some embodiments, to: cause increased insect mortality and / or growth inhibition when insects are exposed to the TVP-insecticide protein compared to TVP alone, increase the expression of the TVP-insecticide protein (e.g., in host cells or expression systems), and / or affect the post-translational processing of the TVP-insecticide protein. In some embodiments, the TVP-insecticide protein may be a polymer comprising two or more TVPs. In some embodiments, the TVP-insecticide protein may be a polymer comprising two or more TVPs, where the TVPs are functionally linked via linker peptides (e.g., cleavable and / or non-cleavable linkers). In some embodiments, the TVP-insecticide protein may refer to one or more TVPs functionally linked to one or more proteins, the one or more proteins being a stabilizing domain (STA), an endoplasmic reticulum signaling protein (ERSP), an insect-cleavable or insect-incapable linker (L), and / or any other combination thereof. In some embodiments, the TVP-insecticide protein may be a non-natural protein comprising (1) a wild-type Ta1b protein and (2) an additional peptide, polypeptide, or protein (e.g., ERSP, linker, STA, UBI, or histidine tag or similar marker).

[0243] In some embodiments, the TVP-insecticide protein may contain one or more TVPs listed in Table 1 (e.g., SEQ ID NOs. 2-15, SEQ ID NOs. 49-53, and SEQ ID NOs. 77-110). In some embodiments, the insecticide protein may contain a TVP homopolymer (e.g., two or more identical TVP monomers). In some embodiments, the insecticide protein may contain a TVP heteropolymer (e.g., two or more different TVP monomers).

[0244] In some embodiments, the TVP-insecticide protein may comprise a homopolymer of two or more TVPs, each with the same amino acid sequence. For example, in some embodiments, the TVPs are R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A , may have a polypeptide comprising the amino acid substitutions R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG, and such polypeptide may have a polypeptide comprising the amino acid substitutions R38AT43A, A8NT43A, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG. The same amino acid substitutions (i.e., R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38) It is linked to another polypeptide containing one or more of the same TVPs having the amino acid substitutions AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0245] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, where the amino acid sequences of each TVP are the same or different. For example, in some embodiments, the TVPs are R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K It may have one polypeptide containing the amino acid substitutions 18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG, and such one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔ G, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0246] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, where the amino acid sequences of each TVP are the same or different. For example, in some embodiments, the TVP may have one polypeptide containing the amino acid substitution R9Q compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1: R9Q, K18A, R38A, A8N, A8S, R9N, T11 P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0247] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution R9QΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0248] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein may comprise, for example, one polypeptide containing the amino acid substitution K18A compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. ru: R9Q, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0249] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution K18AΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0250] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein may comprise, for example, one polypeptide containing the amino acid substitution R38A compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. ru: R9Q, K18A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0251] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein may comprise, for example, one polypeptide containing the amino acid substitution R38AΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide may be linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0252] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution A8N compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. :R9Q, K18A, R38A, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0253] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution A8NΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0254] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution A8S compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. :R9Q, K18A, R38A, A8N, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0255] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution A8SΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0256] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution R9N compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. :R9Q, K18A, R38A, A8N, A8S, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0257] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution R9NΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0258] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein may comprise, for example, one polypeptide containing the amino acid substitution T11P compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. ru: R9Q, K18A, R38A, A8N, A8S, R9N, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0259] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution T11PΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. The following are present: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0260] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein may comprise, for example, one polypeptide containing the amino acid substitution T43A compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. ru: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43A, R9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0261] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution T43AΔG compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. :R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43AR9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0262] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein may comprise, for example, one polypeptide containing the amino acid substitution RQ9 / T43A compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. ru: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43AR9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0263] In some embodiments, the TVP-insecticide protein may comprise a heteropolymer of two or more TVPs, each TVP having different amino acids, and the TVP-insecticide protein comprises, for example, one polypeptide containing the amino acid substitution RQ9 / T43A compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and this one polypeptide is linked to another polypeptide containing one or more of the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, T11PT43AR9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, T11PT43AΔG, and / or any combination thereof.

[0264] In some embodiments, the TVP-insecticide protein may comprise a fusion protein containing two or more TVPs separated by a cleavable or non-cleavable linker, the amino acid sequences of each TVP may be the same or different. For example, in some embodiments, the first TVP polymer may have the following amino acid sequences compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9NΔG, T11PΔG, T43AΔG, R9QT43 The amino acid A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, or T11PT43AR9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG and / or T11PT43AΔG. The first TVP polymer may have substitutions, and the first TVP polymer is fused with a second TVP polymer which may have the following amino acid substitutions compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: R9Q, K18A, R38A, A8N, A8S, R9N, T11P, T43A, R9QΔG, K18AΔG, R38AΔG, A8NΔG, A8SΔG, R9 NΔG, T11PΔG, T43AΔG, R9QT43A, K18AT43A, R38AT43A, A8NT43A, A8ST43A, R9NT43A, or T11PT43AR9QT43AΔG, K18AT43AΔG, R38AT43AΔG, A8NT43AΔG, A8ST43AΔG, R9NT43AΔG, and / or T11PT43AΔG.

[0265] In some embodiments, the insecticidal protein may comprise a fusion protein containing two or more TVPs separated by a cleavable or incleavable linker, the amino acid sequences of each TVP may be the same or different, and the linker may be cleavable in the intestine or hemolymph of an insect. Examples of methods for generating cleavable and incleavable linkers can be found in U.S. Patent Application No. 15 / 727,277 and PCT Application No. PCT / US2013 / 030042, the disclosures of these documents incorporated herein by reference in their entirety.

[0266] In some embodiments, the TVP-insecticide protein may comprise one or more TVPs having amino acid sequences from SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, and SEQ ID NOs: 77-110. In some embodiments, the TVP may include an amino acid sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with the amino acid sequences of SEQ ID NOs. 2-15, SEQ ID NOs. 49-53, or SEQ ID NOs. 77-110.

[0267] In some embodiments, the TVP-insecticide protein may comprise one or more TVPs having amino acid sequences from SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, and SEQ ID NOs: 77-110. In some embodiments, the TVP may include an amino acid sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity with the amino acid sequence "EPDEICRAQMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYAACHEAQKG" (Sequence ID 51).

[0268] Method for generating TVP

[0269] Methods for producing proteins are well known in the art, and various techniques are available. For example, in some embodiments, proteins may be produced using recombinant methods or synthesized chemically.

[0270] In some embodiments, the TVPs of the present invention can be produced using any known method for producing proteins. For example, in some embodiments, but not limited to them, TVPs can be produced using recombinant expression systems (such as yeast or bacterial expression systems). On the other hand, those skilled in the art will recognize that other methods of protein production are also available.

[0271] In some embodiments, the present invention provides a method for generating TVP using a recombinant expression system.

[0272] In some embodiments, the present invention includes, or is essentially derived from, a method for generating TVP, or comprises a method for generating TVP, the method comprising: preparing a vector comprising (a) a first expression cassette comprising, or comprising, a first expression cassette comprising, or comprising (a) a polynucleotide or a complementary nucleotide sequence thereof capable of encoding TVP; (b) introducing the vector into a host cell (e.g., a bacterium or yeast, or an insect, or a plant, or an animal cell); and (c) growing a yeast strain in a growth medium under conditions capable of enabling the expression and secretion of TVP into the growth medium. In some related embodiments, the host cell is a yeast cell.

[0273] The present invention can be implemented in a wide range of host cells (see the Host Cells section below). In fact, end users of the present invention can implement the teachings in any host cell of their choice. Thus, in some embodiments, the host cell can be any host cell that meets the needs of the end user. That is, in some embodiments, TVP expression can be achieved using various host cells according to the teachings herein. For example, in some embodiments, one user may desire to use one particular type of host cell (e.g., yeast cells or bacterial cells), while such host cells may be incompatible with another user, and the range of preferences for a given host cell can range from availability to cost.

[0274] For example, in some embodiments, the present invention includes, or is essentially derived from, a method for generating TVP, or comprises a method for generating TVP, the method comprising: preparing a vector comprising (a) a first expression cassette comprising, or comprising, a first expression cassette comprising, or comprising (a) a polynucleotide or a complementary nucleotide sequence thereof capable of encoding TVP; (b) introducing the vector into a host cell (e.g., a bacterium or yeast, or an insect, or a plant, or an animal cell); and (c) growing a yeast strain in a growth medium under conditions capable of enabling the expression and secretion of TVP into the growth medium. In some relevant embodiments, the host cell is a yeast cell.

[0275] Isolation and mutation introduction of wild-type U1-agatoxin-Ta1b

[0276] TVP can be obtained by creating a mutation in the wild-type U1-agatoxin-Ta1b polynucleotide sequence, inserting the U1-agatoxin-Ta1b variant polynucleotide (tvp) sequence into a suitable vector, transforming the host organism in a manner that expresses the polynucleotide encoding TVP, culturing the host organism to produce the desired amount of TVP, and then purifying the TVP from within and / or around the host organism.

[0277] Inducing mutations in the wild-type U1-agatoxin-Ta1b polynucleotide sequence can be achieved by various means well known to those skilled in the art. Mutation methods include the Kunkel method, cassette mutagenesis, PCR site-directed mutagenesis, "delitto perfetto" method, direct gene deletion and site-directed mutagenesis using PCR and a single reusable marker, direct gene deletion and site-directed mutagenesis using PCR and a single reusable marker with a long homologous region, transplacement, "pop-in pop-out" method, and CRISPR-Cas9.Examples of site-directed mutagenesis include: Ruvkun & Ausubel, A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85-8; Wallace et al., Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981 Aug 11;9(15):3647-56; Dalbadie-McFarland et al., Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci US A. 1982 Nov;79(21):6409-13; Bachman. Site-directed mutagenesis. Methods Enzymol. 2013;529:241-8; 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, all disclosures of the aforementioned references are incorporated herein by reference in their entirety.

[0278] The wild-type U1-agatoxin-Ta1b toxin can be isolated from spider venom. Spider venom can be isolated from the venom glands of spiders (e.g., spiders such as Eratigena agrestis) using any method known to those skilled in the art. For example, in some embodiments, venom can be isolated from spiders according to the method described in U.S. Patent No. 5,688,764, the disclosure of which is incorporated herein by reference in its entirety.

[0279] The wild-type U1-agatoxin-Ta1b polynucleotide sequence can be obtained by screening a genomic library using a primer probe targeting the U1-agatoxin-Ta1b polynucleotide sequence. Alternatively, the wild-type U1-agatoxin-Ta1b polynucleotide sequence and / or TVP polynucleotide sequence can be chemically synthesized. For example, the wild-type U1-agatoxin-Ta1b polynucleotide sequence and / or TVP polynucleotide sequence can be produced using oligonucleotide synthesis methods (such as the phosphoramidite method, triester method, phosphite method, or H-phosphonate method) (see Engels, J. Wand Uhlmann, E. (1989), Gene Synthesis [New Synthetic Methods (77)]. Angew. Chem. Int. Ed. Engl., 28:716-734 (the disclosure of that document is incorporated herein by reference in its entirety)).

[0280] Chemical synthesis of TVP polynucleotides

[0281] In some embodiments, polynucleotide sequences encoding TVP can be chemically synthesized using commercially available polynucleotide synthesis services (such as those provided by GENEWIZ® (e.g., TurboGENE®, PriorityGENE, and FragmentGENE) or SIGMA-ALDRICH® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos)). Examples of methods for generating DNA and chemically custom synthesized polynucleotides are well known in the art and are shown in U.S. Patent No. 5,736,135 (Filing No. 08 / 389,615, filed February 13, 1995), the disclosure of which is incorporated herein by reference in its entirety.Agarwal, et al.,Chemical synthesis of polynucleotides.Angew Chem Int Ed Engl.1972 Jun;11(6):451-9, Ohtsuka et al.,Recent developments in the chemical synthesis of polynucleotides.Nucleic Acids Res.1982 Nov 11;10(21):6553-6570, Sondek & Shortle.A general strategy for random insertion and substitution mutagenesis:substoichiometric coupling of trinucleotide phosphoramidites.Proc Natl Acad Sci US A.1992 Apr 15;89(8):3581-3585, Beaucage SL,et al.,Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach.Tetrahedron,Elsevier Science See also Publishers, Amsterdam, NL, vol.48, No.12, 1992, pp.2223-2311, and Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Methods in Molecular Biology Vol.20. The disclosures in these documents are incorporated herein by reference in their entirety.

[0282] By chemically synthesizing polynucleotides, it becomes possible to generate DNA sequences that produce desired polypeptides based on the arrangement of nucleotides within the sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A], or thymine [T] molecules). The mRNA sequence transcribed from the chemically synthesized DNA polynucleotide can be translated into an amino acid sequence, where each amino acid corresponds to a codon in the mRNA sequence. Therefore, the amino acid composition of the polypeptide chain translated from the mRNA sequence can be modified by altering the underlying codon that determines which of the 20 amino acids is added to the growing polypeptide. Thus, introducing mutations into DNA (such as insertions, substitutions, deletions, and frameshifts) can result in amino acid insertions, substitutions, or deletions depending on the underlying codon.

[0283] Obtaining TVPs from chemically synthesized DNA polynucleotide sequences and / or wild-type DNA polynucleotide sequences modified via mutagenesis can be achieved by cloning the DNA sequence into a suitable vector. A variety of expression vectors, host organisms, and cloning strategies are available and are known to those skilled in the art. For example, a vector may be a plasmid into which a heterologous gene and / or expression cassette can be introduced into yeast cells to produce transcription and translation. The term “vector” is used to refer to a nucleic acid sequence as a carrier nucleic acid molecule into which it can be inserted for the purpose of introduction into a replicable cell. A vector may contain “vector elements,” such as an origin of replication (ORI), a gene conferring antibiotic resistance to enable selection, a multi-cloning site, a promoter region, a selection marker for non-bacterial transfect, and a primer binding site. A nucleic acid sequence may be “foreign,” meaning that such a nucleic acid sequence is foreign to the cell into which the vector is introduced, or that such a sequence is homologous to sequences in the cell but is located in a position within the host cell nucleic acid where such a sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will have sufficient skill to construct vectors via standard recombination techniques. Such recombination techniques are described in Sambrook et al., 1989 and Ausubel et al., 1996, both of which are incorporated herein by reference. In addition to encoding Ta1b variant polynucleotides, vectors can also encode target-directed molecules. Target-directed molecules guide desired nucleic acids to specific tissues, cells, or other locations.

[0284] Vectors and transformations

[0285] In some embodiments, TVP polynucleotides can be cloned into vectors using various cloning strategies and commercially available cloning kits and materials readily available to those skilled in the art. For example, TVP polynucleotides can be cloned into vectors using strategies (such as the SnapFast strategy, Gateway strategy, TOPO strategy, Gibson strategy, LIC strategy, InFusionHD strategy, or Electra strategy). Numerous commercially available vectors exist that can be used to generate TVP. For example, TVP polynucleotides can be generated using polymerase chain reaction (PCR) and then subjected to 5 minutes of conjugation at room temperature with the pCR®II-TOPO vector or the PCR®2.1-TOPO® vector (commercially available as the TOPO®TACloning® kit supplied by Invitrogen). Next, the TOPO® reaction product can be used to transform competent cells, which can then be selected based on their 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. Also see Adams et al. Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997. The disclosures in that document are incorporated herein by reference in their entirety).

[0286] In some embodiments, the polynucleotide encoding TVP can be cloned into a vector (such as a plasmid, cosmid, virus (bacteriophage, animal virus, and plant virus), and / or artificial chromosome (e.g., YAC)).

[0287] In some embodiments, the polynucleotide encoding TVP can be inserted into a vector (e.g., a plasmid vector using E. coli as the host) by: digesting about 2–5 μg of vector DNA with restriction enzymes necessary to make the target DNA segment insertable, then incubating overnight to achieve complete digestion (dephosphorylation of the 5' end may be performed using alkaline phosphatase to avoid autoligation / recirculation), and purifying the digested vector on a gel. The target DNA segment (e.g., the polynucleotide encoding TVP) is then amplified via PCR, and any excess enzymes, primers, unincorporated dNTPs, short failed PCR products, and / or salts from the PCR reaction are removed using methods known to those skilled in the art (e.g., by using a PCR purification kit). The target DNA segment is ligated with the vector by creating a mixture containing approximately 20 ng of vector, approximately 100–1,000 ng of the target DNA segment, 2 μL of 10× buffer (i.e., 30 mM Tris-HCl, 4 mM MgCl2, 26 μM NAD, 1 mM DTT, 50 μg / ml BSA, pH 8 (stored at 25°C)), and 1 μL of T4 DNA ligase (the total volume of all components is increased to 20 μL by adding H2O). The ligation reaction mixture can then be incubated at room temperature for 2 hours or overnight at 16°C. Subsequently, the ligation reaction mixture (i.e., approximately 1 μL) can be used to transform competent cells (e.g., by electroporation or chemical methods), and the vector containing the target DNA segment can be identified by performing colony PCR.

[0288] In some embodiments, the polynucleotide encoding TVP, along with other DNA segments that together constitute a TVP-expressing ORF, can be designed to result in secretion from host yeast cells. An example of how to design a TVP-expressing ORF is as follows: the ORF can begin with a signal peptide sequence, with the DNA sequence encoding the Kex2 cleavage site (lysine-arginine) placed downstream, followed by a TVP polynucleotide transgene with a glycine-serine codon appended to its 5' end, and finally a stop codon appended to its 3' end. These elements are then expressed in yeast cells as a single open reading frame (ORF) to form a fusion peptide. Alpha-conjugation factor (αMF) signal sequences are most commonly used to facilitate the metabolic processing of recombinant insecticidal peptides via the endogenous secretory pathway in recombinant yeast. In other words, the expression fusion peptide typically enters the endoplasmic reticulum, where the α-conjugation factor signal sequence is removed by signal peptidase activity. The resulting insecticidal peptide is then transported to the Golgi apparatus, where the aforementioned lysine-arginine dipeptide is completely removed by Kex2 endoprotease, after which the mature polypeptide (i.e., TVP) is secreted extracellularly.

[0289] In some embodiments, polypeptide expression levels in recombinant yeast cells can be enhanced by optimizing codons based on a specific host yeast species. Codons of native frequency found in the endogenous open reading frame of a given host organism do not necessarily need to be optimized for high-efficiency expression. Furthermore, different yeast species (e.g., Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.) have different codons that are optimal for high-efficiency expression. Therefore, for TVP-expressing ORFs, codon optimization should be considered, including the sequence elements encoding the signal sequence, the sequence elements encoding the Kex2 cleavage site, and the sequence elements encoding TVP, because these sequence elements are initially translated as a single fusion peptide in recombinant yeast cells.

[0290] In some embodiments, codon-optimized TVP expression ORFs can be ligated into yeast-specific expression vectors for yeast expression. Many expression vectors are available for yeast expression, including episomal and embedded vectors, and these are typically designed for specific yeast strains. The appropriate expression vector should be carefully selected in light of the specific yeast expression system to be used for peptide production. In some embodiments, embedded vectors can be used that are integrated into the chromosomes of the transformed yeast cells and remain stable throughout the cell division and proliferation cycle. The embedded DNA sequence is homologous to the target genomic DNA locus in the transformed yeast species, and such embedded sequences include pLAC4, 25S rDNA, pAOX1, and TRP2. The insecticidal peptide transgene may be located adjacent to the embedded DNA sequence (insertion vector) or within the embedded DNA sequence (substitution vector).

[0291] In some embodiments, the expression vector may include E. coli elements for preparing DNA in E. coli, such as E. coli origins and antibiotic selection markers. In some embodiments, the vector may include an array of sequence elements necessary for the expression of the target transgene, such as transcription promoters, terminators, yeast selection markers, and integrated DNA sequences homologous to host yeast DNA. Many suitable yeast promoters are available, including natural and engineered promoters, such as pLAC4, pAOX1, pUPP, pADH1, pTEF, and pGal1, and in some embodiments, such yeast promoters may be used.

[0292] In some embodiments, selection methods such as acetamide prototrophic selection, zeosin resistance selection, genethecin resistance selection, noseoslysin resistance selection, uracil deficiency selection, and / or other selection methods may be used. For example, in some embodiments, the Aspergillus nidulans amdS gene may be used as a selectable marker. Examples of methods for using selectable markers can be found in U.S. Patent No. 6,548,285 (filed April 3, 1997), No. 6,165,715 (filed June 22, 1998), and No. 6,110,707 (filed January 17, 1997), the disclosures of these documents are incorporated herein by reference in their entirety.

[0293] In some embodiments, the polynucleotide encoding TVP can be inserted into the pKLAC1 plasmid. pKLAC1 is commercially available from New England Biolabs® Inc. (Catalog No. (NEB #E1000)). pKLAC1 is designed to achieve high levels of recombinant protein (e.g., TVP) expression 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 restriction enzyme or BstXI restriction enzyme and has an MCS downstream of the αMF secretory signal. The αMF secretory signal leads the recombinant protein into the secretory pathway, where it is subsequently cleaved via Kex2, resulting in the target peptide (e.g., TVP). Kex2 is a calcium-dependent serine protease involved in the activation of the proprotein in the secretory pathway and is commercially available (PeproTech®; Catalog No. 450-45).

[0294] In some embodiments, a polynucleotide encoding TVP may be inserted into the pKlac1 plasmid or subcloned into the pKlac1 plasmid, after which selection of yeast colonies transformed with the TVP-encoding polynucleotide-ligated pKLAC1 plasmid may be performed. Yeast (e.g., K. lactis) transformed with the TVP-encoding polynucleotide-ligated pKLAC1 plasmid can be selected based on acetamidase (amdS), which enables the transformed yeast cells to grow in YCB medium containing acetamide as the sole nitrogen source. Once positivity is confirmed, yeast colonies transformed with the TVP-encoding polynucleotide-ligated pKLAC1 plasmid are identified.

[0295] In some embodiments, the polynucleotide encoding the TVP can be inserted into other commercially available plasmids and / or vectors readily available to those skilled in the art. For example, plasmids are available from Addgene (a non-profit plasmid repository), GenScript®, Takara®, Qiagen®, and Promega®.

[0296] In some embodiments, yeast cells transformed with one or more TVP expression cassettes produce TVP in the yeast culture, and the TVP yield per liter of medium is at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, and at least 190 mg / L. g / L, 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / 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 / It may be 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.

[0297] In some embodiments, one or more expression cassettes containing polynucleotides capable of expressing TVP are inserted into the vector, resulting in TVP yields per liter of medium (supernatant of yeast fermentation broth) of approximately 100 mg / L to 100,000 mg / L, approximately 110 mg / L to 100,000 mg / L, approximately 120 mg / L to 100,000 mg / L, approximately 130 mg / L to 100,000 mg / L, approximately 140 mg / L to 100,000 mg / L, approximately 150 mg / L to 100,000 mg / L, approximately 160 mg / L to 100,000 mg / L, Approx. 170mg / L~Approx. 100,000mg / L, Approx. 180mg / L~Approx. 100,000mg / L, Approx. 190mg / L~Approx. 100,000mg / L, Approx. 200mg / L~Approx. 100,000mg / L, Approx. 250mg / L~Approx. 100,000mg / L, Approx. 500mg / L~Approx. 100,0 00mg / 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 250 0mg / L~Approx. 100,000mg / L, Approx. 3000mg / L~Approx. 100,000mg / L, Approx. 3500mg / L~Approx. 100,000mg / L, Approx. 4000mg / L~Approx. 100,000mg / L, Approx. 4500mg / L~Approx. 100,000mg / L, Approx. 5000mg / L~Approx. 100 ,000mg / L, approx. 5500mg / L~approx. 100,000mg / L, approx. 6000mg / L~approx. 100,000mg / L, approx. 6500mg / L~approx. 100,000mg / L, approx. 7000mg / L~approx. 8000mg / L~100,000mg / L, 8500mg / L~100,000mg / L, 9000mg / L~100,000mg / L, 9500mg / L~100,000mg / L, 10000mg / L~100,000mg / L, 10500mg / L ~100,000mg / L, 11000mg / L~100,000mg / L, 11500mg / L~100,000mg / L, 12000mg / L~100,000mg / L, 12500mg / L~100,000mg / L, 13000mg / L~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 99000m, The concentration can range from g / L to approximately 100,000 mg / L, or from approximately 99,500 mg / L to approximately 100,000 mg / L.

[0298] In some embodiments, one or more expression cassettes containing polynucleotides capable of expressing TVP are inserted into the vector, resulting in TVP yields per liter of medium (supernatant of yeast fermentation broth) of approximately 100 mg / L to 100,000 mg / L, approximately 100 mg / L to 99,500 mg / L, approximately 100 mg / L to 99,000 mg / L, approximately 100 mg / L to 98,500 mg / L, approximately 100 mg / L to 98,000 mg / L, approximately 100 mg / L to 97,500 mg / L, approximately 100 mg / L to 97,000 mg / L, and approximately 100 mg / L to 9 6500mg / L, about 100mg / L to about 96000mg / L, about 100mg / L to about 95500mg / L, about 100mg / L to about 95000mg / L, about 100mg / L to about 94500mg / L, about 100mg / L to about 94000mg / L, about 100mg / L to about 93500m g / L, approx. 100 mg / L ~ approx. 93000 mg / L, approx. 100 mg / L ~ approx. 92500 mg / L, approx. 100 mg / L ~ approx. 92000 mg / L, approx. 100mg / L~Approx. 90000mg / L, Approx. 100mg / L~Approx. 89500mg / L, Approx. 100mg / L~Approx. 89000mg / L, Approx. 100mg / L~Approx. 88500mg / L, Approx. 100mg / L~Approx. 88000mg / L, Approx. g / L ~ approx. 87000 mg / L, approx. 100 mg / L ~ approx. 86500 mg / L, approx. 100 mg / L ~ approx. 86000 mg / L, approx. 100 mg / L ~ approx. 85500 mg / L, approx. 100 mg / L ~ approx. 84000mg / 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 81000 mg / 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.It can range from approximately 100 mg / L to approximately 2500 mg / L, approximately 100 mg / L to approximately 2000 mg / L, approximately 100 mg / L to approximately 1500 mg / L, approximately 100 mg / L to approximately 1000 mg / L, approximately 100 mg / L to approximately 1000 mg / L, approximately 100 mg / L to approximately 750 mg / L, approximately 100 mg / L to approximately 500 mg / L, approximately 100 mg / L to approximately 250 mg / L, approximately 100 mg / L to approximately 100 mg / L, or approximately 100 mg / L to approximately 110 mg / L.

[0299] In addition to the DNA polynucleotide sequence encoding TVP, additional DNA segments known as regulatory elements, capable of enhancing the expression of foreign DNA or transgenes, can be cloned into the vector. Examples of such additional DNA segments include (1) promoter, terminator, and / or enhancer elements, (2) appropriate mRNA-stabilizing polyadenylation signals, (3) intrasequence ribosome entry sites (IRESs), (4) introns, and (5) post-transcriptional regulatory elements. The combination of the target DNA segment (e.g., tvp) and any one of the aforementioned cis-acting elements is called an "expression cassette."

[0300] A single expression cassette may include one or more of the aforementioned regulatory elements and a polynucleotide capable of expressing TVP. For example, in some embodiments, a TVP expression cassette may include a polynucleotide capable of expressing TVP, as well as an α-MF signal, a Kex2 site, an LAC4 terminator, an ADN1 promoter, and an acetamidase (amdS) selection marker (adjacent to the LAC4 promoter at the 5' and 3' ends).

[0301] In some embodiments, there may be multiple expression cassettes cloned into the vector. For example, in some embodiments, there may be a first expression cassette containing a polynucleotide capable of expressing TVP. In alternative embodiments, there may be two expression cassettes capable of encoding TVP (i.e., a dual expression cassette). In other embodiments, there may be three expression cassettes capable of encoding TVP (i.e., a triple expression cassette).

[0302] In some embodiments, a dual expression cassette can be generated by subcloning a second TVP expression cassette into a vector containing a first TVP expression cassette.

[0303] In some embodiments, a triple expression cassette can be generated by subcloning a third TVP expression cassette into a vector containing a first TVP expression cassette and a second TVP expression cassette.

[0304] In some embodiments, yeast cells transformed with one or more TVP expression cassettes produce TVP in the yeast culture, and the TVP yield per liter of yeast culture medium is at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, at least 100 mg / L, at least 110 mg / L, at least 120 mg / L, at least 130 mg / L, at least 140 mg / L, at least 150 mg / L, at least 160 mg / L, at least 170 mg / L, at least 180 mg / L, and at least 1 90 mg / L, 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 11,000 mg / 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 It may be 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.

[0305] In some embodiments, one or more expression cassettes containing polynucleotides capable of expressing TVP are inserted into a vector (e.g., pKlac1 plasmid), resulting in a TVP yield (supernatant of yeast fermentation broth) of approximately 100 mg / L. For example, in some embodiments, two expression cassettes containing polynucleotides capable of expressing TVP are inserted into a vector (e.g., pKS482 plasmid), resulting in a TVP yield (supernatant of yeast fermentation broth) of approximately 2 g / L. Alternatively, in some embodiments, three expression cassettes containing polynucleotides capable of expressing TVP may be inserted into a vector (e.g., pKlac1T plasmid).

[0306] In some embodiments, multiple TVP expression cassettes can be transfected into yeast to allow the incorporation of one or more copies of an optimized TVP transgene into the K. lactis genome. An example of a method for introducing multiple TVP expression cassettes into the K. lactis genome is as follows: Synthesize a TVP expression cassette DNA sequence containing an intact LAC4 promoter element, a codon-optimized TVP expression ORF element, and a pLAC4 terminator element. Ligate this intact expression cassette with the pKlac1 vector at a position between the SalI restriction enzyme recognition site and the KpnI restriction enzyme recognition site (downstream of the pLAC4 terminator in pKS477) to obtain a dual transgene TVP expression vector (pKS482). Subsequently, this dual transgene vector (pKS482) is linearized using SacII restriction endonuclease and transformed into the K. lactis YCT306 strain by electroporation. Next, the obtained yeast colonies are grown on a YCB agar plate supplemented with 5 mM acetamide. Only acetamidase-expressing cells can efficiently utilize acetamide as a metabolic nitrogen source. Approximately 100 to 400 colonies can be picked from a pKS482 yeast plate to evaluate the yeast colonies. Seeds derived from the colonies are cultured in 2.2 mL of normal K. lactis medium supplemented with 2% sugar alcohol as a carbon source. The cultures are incubated at 23.5°C for 6 days with shaking at 280 rpm, at which point the cell density in the culture (indicated by absorbance at 600 nm (OD600)) reaches its maximum level. Next, cells are 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 pore size membrane for HPLC yield analysis.

[0307] In some embodiments, the vector comprises a polynucleotide capable of encoding a TVP having an amino acid sequence that is 90% similar to any one of the sequences shown in SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0308] In other embodiments, the vector comprises a polynucleotide having an amino sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150, or a complementary nucleotide sequence thereof.

[0309] Chemical synthesis of TVP

[0310] The production of TVP may involve peptide synthesis or the chemical synthesis of peptides and / or polypeptides. These methods can be carried out by those skilled in the art and / or by using 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).

[0311] In some embodiments, peptide synthesis can generally be achieved using a strategy of coupling the carboxyl group of a subsequent amino acid to the N-terminus of a preceding amino acid to generate a nascent polypeptide chain, which is the reverse process of naturally occurring polypeptide synthesis.

[0312] Peptide deprotection is a crucial first step in the chemical synthesis of polypeptides. Peptide deprotection is the process of preventing the functional groups of an amino acid from being involved in undesirable or nonspecific reactions or side reactions by blocking the reactive groups of that amino acid using chemicals. In other words, the amino acid is "protected" from being involved in these undesirable reactions.

[0313] Prior to peptide chain synthesis, amino acids must be "deprotected" to enable chain formation (i.e., amino acid bonding). Chemicals used for N-terminus protection include 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc), which can be removed using a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoroacetic acid (TFA)), respectively.

[0314] The required C-terminal protecting group 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, as the solid support acts as the protecting group. Several different protecting groups are required for the side-chain amino acids, and these groups 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.

[0315] In peptide synthesis procedures, amino acid coupling is the next step. To achieve amino acid coupling, the C-terminal carboxylic acid of the introduced amino acid must be activated. This activation can be achieved using a carbodiimide (such as diisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC)), which reacts with the carboxyl group of the introduced amino acid to form an O-acylisourea intermediate. Subsequently, the O-acylisourea intermediate is replaced by nucleophilic attack via the primary amino group on the N-terminus of the growing peptide chain. This reactive intermediate produced by the carbodiimide can cause racemization of the amino acid. To avoid racemization of the amino acid, a reagent (such as 1-hydroxybenzotriazole (HOBt)) is added to react with the O-acylisourea intermediate. Other coupling agents that may be used include 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), which are used in conjunction with additional activating bases. Finally, after the deprotection and coupling of the amino acids,

[0316] At the end of the synthesis process, protecting groups must be removed from the polypeptide. This removal is typically carried out via acid digestion. Determining which reagents are needed for peptide cleavage depends on the protection scheme and total synthesis method used. For example, in some embodiments, the Bzl and Boc groups can be cleaved using hydrogen bromide (HBr), hydrogen fluoride (HF), or trifluoromethanesulfonic acid (TFMSA). Alternatively, in other embodiments, a weak acid (such as TFA) may result in acid digestion of the tBut and Fmoc groups. Finally, the peptide can be purified based on its physiological and chemical characteristics (e.g., charge, size, hydrophobicity, etc.). Techniques that may be used for peptide purification include reversed-phase chromatography (RPC), size exclusion chromatography, partition chromatography, high-performance liquid chromatography (HPLC), and ion-exchange chromatography (IEC).

[0317] Examples of peptide synthesis methods include Anderson G.W. and McGregor A.C. (1957) T-butyloxycarbonyl amino acids and their use in peptide synthesis. Journal of the American Chemical Society. 79, 6180-3, Carpino L.A. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1,1-disubstituted-2-arenesulfonhydrazides 1-4. Journal of the American Chemical Society. 79, 4427-31, McKay F.C. and Albertson N.F. (1957) New amine-masking groups for peptide synthesis. Journal of the American Chemical Society. 79, 4686-90, Merrifield R.B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 85, 2149-54, Carpino L.A. and Han G.Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry. 37, 3404-9, and A Lloyd-Williams P. et al. (1997) Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press.These can be found in U.S. Patent Nos. 278, 3,714,140 (filed March 16, 1971), 4,411,994 (filed June 8, 1978), 7,785,832 (filed January 20, 2006), 8,314,208 (filed February 10, 2006), and 10,442,834 (filed October 2, 2015), as well as U.S. Patent Application No. 2005 / 0165215 (filed December 23, 2004), the disclosures of which are incorporated herein by reference in their entirety.

[0318] Example of a method

[0319] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprising the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising at least one amino acid substitution compared to, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions that allow it to function to enable TVP expression.

[0320] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1 (b) preparing a vector comprising X1 being A, S, or N, X2 being R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 being T or P, X4 being K or A, X5 being R or A, Z1 being T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 being K or absent, and X7 being G or absent, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP has one amino acid substitution in X1, X2, X3, X4, or X5.

[0321] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, X (b) preparing a vector in host cells and (c) growing host cells in growth medium under conditions functional to enable TVP expression, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent, (b) introducing the vector into host cells, and (c) growing host cells in growth medium under conditions functional to enable TVP expression, wherein TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is glycine.

[0322] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1 (b) preparing a vector in host cells, and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is absent.

[0323] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and X1 (b) preparing a vector in host cells and (c) growing host cells in growth medium under conditions functional to enable TVP expression, wherein X1, X2, X3, X4, or X5 has one amino acid substitution and X6 and X7 are absent.

[0324] In some embodiments, a method for producing TVP or TVP-insecticidal protein is (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising 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, (b) preparing a vector in host cells and (c) growing host cells in growth medium under conditions functional to enable TVP expression, wherein TVP comprises an amino sequence shown in any one of S, 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, (b) introducing the vector into host cells, and (c) growing host cells in growth medium under conditions functional to enable TVP expression, wherein TVP comprises an amino sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0325] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, (b) preparing a vector in host cells and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NOs: 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, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150.

[0326] In some embodiments, a method for producing TVP or TVP-insecticidal protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprising at least one amino acid sequence compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a plasmid containing an alpha-MF signaling plasmid, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions that allow for the expression of TVP, wherein the vector is a plasmid containing an alpha-MF signaling plasmid.

[0327] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector such that 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, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions that enable the expression of TVP, wherein the alpha-MF signal is functional to express the alpha-MF signal peptide.

[0328] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide being at least compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising one amino acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein the vector is transformed into host cells.

[0329] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide being at least 1 (b) preparing a vector comprising one amino acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein the host cells are eukaryotic or prokaryotic cells.

[0330] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide having less than the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising: (a) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions that enable the expression of TVP, wherein the host cells are yeast cells.

[0331] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, Q, N, A, G, N (b) preparing a vector in host cells, and (c) growing the host cells in growth medium under conditions that allow TVP expression to occur, wherein the host cells are yeast cells selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces.

[0332] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide being at least compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising one amino acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein the host cells are Kluyveromyces lactis or Kluyveromyces marxianus.

[0333] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide is less than the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising one amino acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP is secreted into the growth medium.

[0334] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and X1 (b) preparing a vector such that X2 is A, S, or N, X3 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X4 is T or P, X5 is K 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; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions that allow TVP expression to occur, the TVP being secreted into the growth medium, and the TVP being ligated to function with an alpha-MF signal peptide.

[0335] In some embodiments, a method for producing TVP or TVP-insecticidal protein is to (a) prepare a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, and the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the poly The lipeptide is prepared such that it contains at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N; 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. (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions that enable TVP expression, wherein the TVP yield per liter of yeast culture medium is 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, and at least 170 mg / L, at least 180 mg / L, at least 190 mg / L, 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,It will be 500 mg / L, at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,000 mg / L, at least 12,500 mg / L, at least 15,000 mg / L, at least 17,500 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, or at least 100,000 mg / L.

[0336] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1 (b) preparing a vector in host cells, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein the TVP expression results in a TVP yield of at least 100 mg / L per liter of medium.

[0337] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprising at least one amino acid compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, resulting in the expression of TVP in the medium, thereby expressing a single TVP in the medium.

[0338] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and X1 is (b) preparing a vector such that 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; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions that allow TVP expression to occur, resulting in the expression of TVP in the medium, which in turn results in the expression of a TVP fusion polymer containing two or more TVP polypeptides in the medium.

[0339] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, (b) preparing a vector in host cells and (c) growing the host cells in growth medium under conditions that enable the expression of TVP, wherein the vector comprises two or three expression cassettes, each expression cassette being functional to encode TVP of the first expression cassette.

[0340] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X (b) preparing a vector such that X3 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X4 is T or P, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions that allow the vector to function to enable TVP expression, wherein the vector comprises two or three expression cassettes, each expression cassette being functional to encode TVP of a first expression cassette or TVP of a different expression cassette.

[0341] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, the polypeptide comprising at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N (b) preparing a vector in host cells and (c) growing the host cells in growth medium under conditions that enable TVP expression, wherein 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, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions that enable TVP expression, wherein the expression cassette is functional to encode TVP as shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0342] In some embodiments, a method for producing TVP or TVP-insecticide protein is (a) preparing a vector comprising a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, or N, and X2 is R, (b) preparing a vector in host cells and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NOs: 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, (b) introducing the vector into host cells, and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150.

[0343] In some embodiments, a method for producing TVP or TVP-insecticide protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprising at least one argon compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. (b) preparing a vector comprising a amino acid substitution, wherein X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, and X7 is G or absent; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein if Z1 is T or S, TVP is glycosylated.

[0344] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression.

[0345] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, and the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling the expression of TVP, wherein if Z1 is T, the TVP is glycosylated.

[0346] In some embodiments, a method for producing TVP or TVP-insecticidal protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein X1 is Q and Z1 is A.

[0347] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, and the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling the expression of TVP, wherein the TVP comprises the amino sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 49, or SEQ ID NO: 51.

[0348] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence functional to encode TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable expression of TVP, wherein the TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 54, or SEQ ID NO: 56.

[0349] In some embodiments, a method for producing TVP or TVP-insecticidal protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the vector is a plasmid containing an alpha-MF signal.

[0350] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence functional to encode TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein the alpha-MF signal is functional to express an alpha-MF signal peptide.

[0351] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the vector is transformed into host cells.

[0352] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the host cells are eukaryotic or prokaryotic cells.

[0353] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in a growth medium under conditions capable of enabling TVP expression, wherein the host cells are yeast cells.

[0354] In some embodiments, a method for producing TVP or TVP-insecticidal protein is to (a) prepare a vector comprising a first expression cassette, the first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, the TVP comprising an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprising U1-agatoxin as shown in SEQ ID NO: 1. (b) preparing a vector comprising at least one amino acid substitution compared to the wild-type sequence of syn-Ta1b, wherein X1 is R or Q and Z1 is T or A; (c) introducing the vector into host cells; and (d) growing the host cells in growth medium under conditions that allow for the expression of TVP, wherein the host cells are yeast cells selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces.

[0355] In some embodiments, a method for producing TVP or TVP-insecticidal protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the host cells are Kluyveromyces lactis or Kluyveromyces marxianus.

[0356] In some embodiments, a method for producing TVP or TVP-insecticidal protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling the expression of TVP, wherein the TVP is secreted into the growth medium.

[0357] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, and the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the TVP is ligated to function with an alpha-MF signal peptide.

[0358] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, and the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the TVP expression results in a TVP yield per liter of yeast culture medium of at least 70 mg / L, at least 80 mg / L, at least 90 mg / L, and at least 1 00 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, 200 mg / L, at least 500 mg / L, at least 750 mg / L, at least 1,000 mg / L, at least 1,250 mg / L, at least 1,500 mg / L, at least 1,750 mg / L, at least 2,000 mg / L, less At least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, at least 5,000 mg / L, at least 5,500 mg / L, at least 6,000 mg / L, at least 6,500 mg / L, at least 7,000 mg / L, at least 7,500 mg / L, at least 8,000 mg / L, at least 8,500 mg / L, at least 9,000 mg / L, at least 9,500 mg / L, at least 10,The levels will be 000 mg / L, at least 12,500 mg / L, at least 15,000 mg / L, at least 17,500 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, or at least 100,000 mg / L.

[0359] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or complementary nucleotide sequence functional to encode TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable TVP expression, wherein the TVP expression results in a TVP yield of at least 100 mg / L per liter of medium.

[0360] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, resulting in the expression of TVP in the medium, thereby expressing a single TVP in the medium.

[0361] In some embodiments, a method for producing TVP or a TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in a growth medium under conditions capable of enabling TVP expression, thereby expressing TVP in the medium, resulting in the expression of TVP in the medium, and consequently, a TVP fusion polymer comprising two or more TVP polypeptides being expressed in the medium.

[0362] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence functional to encode TVP, and the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable the expression of TVP, wherein the vector comprises two or three expression cassettes, each expression cassette functional to encode the TVP of the first expression cassette or the TVP of a different expression cassette.

[0363] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence functional to encode TVP, and the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable the expression of TVP, wherein the vector comprises two or three expression cassettes, each expression cassette functional to encode the TVP of the first expression cassette or the TVP of a different expression cassette.

[0364] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein the expression cassette is capable of encoding TVP shown in any one of SEQ ID NO: 2, SEQ ID NO: 49, or SEQ ID NO: 51.

[0365] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence functional to encode TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions functional to enable expression of TVP, wherein the TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 54, or SEQ ID NO: 56.

[0366] In some embodiments, a method for producing TVP or TVP-insecticide protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, and the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, and the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling the expression of TVP, wherein if Z1 is T, the TVP is glycosylated.

[0367] In some embodiments, a method for producing TVP or TVP-insecticidal protein comprises (a) preparing a vector comprising a first expression cassette, wherein the first expression cassette comprises a polynucleotide or a complementary nucleotide sequence capable of encoding TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A; (b) introducing the vector into host cells; and (c) growing the host cells in growth medium under conditions capable of enabling TVP expression, wherein X1 is Q and Z1 is A.

[0368] Cell culture and transformation techniques

[0369] The terms “transformation” and “transfect” both describe the process of introducing foreign and / or heterologous DNA or RNA into a host organism. Generally, those skilled in the art may use the term “transformation” to describe the process of introducing foreign and / or heterologous DNA or RNA into bacterial cells, and the term “transfect” to describe the process of introducing foreign and / or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the terms “transformation” and “transfect” are used synonymously regardless of whether the target organism to which the foreign and / or heterologous DNA or RNA described by the process is a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plant, or animal).

[0370] In some embodiments, host cells may be transformed using the following methods: electroporation, cell squeezing, microinjection, impalefection, hydrostatic pressure, sonoporation, phototransfect, continuous injection, lipofection, by using viruses (such as adenoviruses, adeno-associated viruses, lentiviruses, herpes simplex viruses, and retroviruses), chemical phosphate methods, endocytosis via DEAE-dextran or polyethyleneimine (PEI), protoplast fusion, hydrodynamic delivery, magnetofection, nucleoinfection, and / or other methods. Examples of methods relating to transfect and / or transformation techniques can be found in Makrides (2003), Gene Transfer and Expression in Mammalian Cells, Elvesier; Wong, TK & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584-587 (1982), Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3, and Kim & Eberwine, Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010 Aug; 397(8): 3173-3178, and each of these references is incorporated herein by reference in its entirety.

[0371] Electroporation is a technique that applies electricity to cells to make the cell membrane permeable, thereby enabling the introduction of foreign DNA into the cells. Electroporation is well known to those skilled in the art, and the tools and devices necessary to achieve it are commercially available (e.g., Gene Pulser Xcell® Electroporation Systems, Bio-Rad®, Neon® Transfection System for Electroporation, Thermo-Fisher Scientific, and other tools and / or devices). Examples of electroporation methods are shown in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER:Unit-9.3; Saito (2015) Electroporation Methods in Neuroscience. Springer Press; and Pakhomov et al., (2017) Advanced Electroporation Techniques in Biology and Medicine. Taylor & Francis, the disclosures of these documents are incorporated herein by reference in their entirety.

[0372] In some embodiments, electroporation can be used to introduce a vector containing polynucleotides encoding TVP into yeast. For example, for cloning TVP into the pKlac1 plasmid and transforming it into K. lactis cells by electroporation, a suitable yeast species (e.g., Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc.) is seeded in approximately 10-200 mL of yeast expeptone dextrose (YEPD), and the yeast culture is in the early logarithmic phase (e.g., approximately 0.6-2 × 10⁻¹⁶). 8Incubate on a shaker at 30°C until the number of cells / mL is reduced, collect the yeast in a sterile centrifuge tube, centrifuge at 4°C and 3000 rpm for 5 minutes (Note: keep the cells cool during this procedure), wash the cells with 40 mL of ice-cold sterile deionized water, pellet the cells at 23,000 rpm for 5 minutes, repeat this washing step, add 20 mL of 1 M fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol) After resuspending the cells in (hydrolyzed hydrogenated starch, isomalt, lactitol, maltitol, mannitol, and xylitol), they were allowed to settle at 3,000 rpm for 5 minutes, and then resuspended in an appropriate volume of 1 M ice-cold fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol, hydrolyzed hydrogenated starch, isomalt, lactitol, maltitol, mannitol, and xylitol) to a final cell density of 3 × 10⁻⁶. 9 This can be achieved by mixing approximately 1-4 μl (approximately 1 μg) of vector containing the linear polynucleotide encoding TVP with 40 μl of yeast suspension in a 0.2 cm pre-cooled electroperforated cuvette (note: ensure that both sides of the aluminum cuvette are in contact with the sample) at a cell / mL concentration, applying a single pulse at 2000 V to optimize the RC circuit time constant (5 msec), then collecting the cells in 0.5 ml of YED and 0.5 mL of a 1 M fermentable sugar (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohol (e.g., erythritol, hydrolyzed hydrogenated starch, isomalt, lactitol, maltitol, mannitol, and xylitol) mixture, and spreading them on a selection plate.

[0373] In some embodiments, electroporation can be used to introduce a vector containing a polynucleotide encoding TVP into a plant protoplast. For this introduction, sterile plant material is incubated in a protoplast solution (for example, about 8 mL of a solution containing 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 the mixture is placed in a rotary shaker and shaken at 30°C for about 3-6 hours to produce a protoplast. Debris is removed by filtration through a mesh nylon screen with a pore size of 80 μm, the screen is rinsed with approximately 4 ml of plant electroporation buffer (e.g., 5 mM CaCl2, 0.4 M mannitol, and PBS), the protoplasts are integrated into a 15 mL sterile conical centrifuge tube, and then centrifuged at approximately 300 × g for approximately 5 minutes. After centrifugation, the supernatant is discarded and washed with 5 mL of plant electroporation buffer, and the number of protoplasts per mL of liquid is approximately 1.5 × 10⁶. 6 ~2×10 6 The protoplasts are resuspended in plant electroporation buffer to form individual cells, approximately 0.5 mL of the protoplast suspension is transferred to one or more electroporation cuvettes, placed on ice, and the vector (Note: For stable transformation, the vector should be linearized using one of the restriction enzyme methods described above, and approximately 1–10 μg of vector may be used. For transient expression, the vector may be kept in its supercoiled state, and approximately 10–40 μg of vector may be used) is added, the vector and protoplast suspension are mixed, the cuvette is placed in an electroporation apparatus, and it is subjected to one or more shocks at approximately 1–2 kV (a capacitance of 3–25 μF may be used initially during reaction optimization), the cuvette is returned to ice, the transformed cells are diluted 20-fold with complete medium, and the protoplasts are collected after approximately 48 hours.

[0374] host cell

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

[0376] In some embodiments, the host cells used to produce TVP or TVP-insecticidal proteins are prokaryotes. For example, in some embodiments, the host cells may be archaea or bacteria (such as Gram-negative or Gram-positive organisms). Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, 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 TVP or TVP-insecticidal protein may be unicellular. For example, in some embodiments, the host cell may be a bacterial cell (such as a Gram-positive bacterium).

[0378] In some embodiments, the host cell can be a bacterium selected from the group consisting of the following genera: 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、またはThermus。

[0379] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal protein 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, Pseudomonas filiscindens, Pseudomonasfrederiksbergensis, 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、Pseudomonas alcaligenes、Pseudomonasanguilliseptica, 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, Pseudomonas mosselii, Pseudomonasoryzihabitans, 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 cells used to produce TVP or TVP-insecticidal proteins may be eukaryotic.

[0381] In some embodiments, the host cells used to produce TVP or TVP-insecticidal proteins may be cells belonging to the following clades: Opisthokonta, Viridiplantae (e.g., algae and plants), Amebozoa, Cercozoa, Alveolata, Marine flagellates, Heterokonta, Discicristata, or Excavata.

[0382] In some embodiments, the procedures and methods described herein may be achieved using host cells (e.g., metazoans, choanoflagellates, or fungi).

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

[0384] In some embodiments, the procedures and methods described herein may be achieved using host cells that are fungi 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 may be achieved using a host cell that is a fungus belonging to one of the following species: Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces uvarum, Aspergillus flavus, A. terreus, A. awamori, Cladosporium elatum, Cl. Herbarum, Cl. Sphaerospermum, and Cl. Cladosporioides, Magnaporthe grise, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

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

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

[0388] In some embodiments, the host cell used for the production of TVP or TVP-insecticidal protein may be a member of the family Saccharomycetaceae. For example, in some embodiments, the host cell may be one of the following genera within the family Saccharomycetaceae: 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 for the production of TVP or TVP-insecticidal protein may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

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

[0391] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal proteins may be species within the genus Kluyveromyces. For example, the host cells may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.

[0392] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal proteins may be species within the genus Pichia. For example, the host cells may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.

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

[0394] In some embodiments, the host cell used for the production of TVP or TVP-insecticidal protein may be one of the following: Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, or Hansenula anomala.

[0395] Using yeast cells as a host organism to produce recombinant TVP is a special method known to those skilled in the art. In some embodiments, the methods and compositions described herein can be carried out using any yeast species, including, but not limited to, any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces, and any species of the genus Saccharomyces, such as Saccharomyces cerevisiae species selected from, for example, the following strains: INVSc1, YNN27, S150-2B, W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A, and BJ3505. In some embodiments, the members of the genus Pichia include any species of the genus Pichia, such as Pichia pastoris, which is selected from the following strains: Bg08, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, KM71, MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any pep4 knockout strain and any prb1 knockout strain, as well as Pichia selected from the following strains pastoris: Bg08, X-33, SMD1168, and KM71. In some embodiments, any Kluyveromyces species, including any Kluyveromyces species (e.g., Kluyveromyces lactis), may be used to achieve the methods described herein.In addition to the Kluyveromyces lactis species selected from GG799, YCT306, and NRRL Y-1140, the inventors teach that the strains of Kluyveromyces lactis can be selected from the following strains, although selection is not necessarily limited to these 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.

[0396] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal proteins may be Aspergillus oryzae.

[0397] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal proteins may be Aspergillus japonicas.

[0398] In some embodiments, the host cell used for the production of TVP or TVP-insecticidal protein may be Aspergillus niger.

[0399] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal proteins may be Bacillus licheniformis.

[0400] In some embodiments, the host cells used for the production of TVP or TVP-insecticidal proteins may be Bacillus subtilis.

[0401] In some embodiments, the host cell used for the production of TVP or TVP-insecticidal protein may be Trichoderma reesei.

[0402] In some embodiments, the procedures and methods described herein may be achieved using a host cell that is a yeast. Such yeasts include, but are not limited to, any species of the genus Hansenula, and such species are preferably Hansenula polymorpha. In some embodiments, the procedures and methods described herein may be achieved using any yeast species. Such yeast species include, but are not limited to, any species of the genus Yarrowia (e.g., Yarrowia lipolytica). In some embodiments, the procedures and methods described herein may be achieved using any yeast species. Such yeast species include, but are not limited to, any species of the genus Schizosaccharomyces, and such species are preferably Schizosaccharomyces pombe.

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

[0404] The preparation methods for yeast cell fermentation media and stocks are as follows: (1) MSM medium preparation: 2 g / L sodium citrate dihydrate, 1 g / L calcium sulfate dihydrate (0.79 g / L anhydrous calcium sulfate), 42.9 g / L potassium dihydrogen phosphate, 5.17 g / L ammonium sulfate, 14.33 g / L potassium sulfate, 11.7 g / L magnesium sulfate heptahydrate, 2 mL / L PTM1 trace salt solution, 0.4 ppm biotin (derived from 500 × 200 ppm stock), 1-2% pure glycerol or other carbon source. (2) PTM1 trace salt solution: 6.0g of copper sulfate·5H2O, 0.08g of sodium iodide, 3.0g of manganese sulfate·H2O, 0.2g of sodium molybdate·2H2O, 0.02g of boric acid, 0.5g of cobalt chloride, 20.0g of zinc chloride, 65.0g of ferrous sulfate·7H2O, 0.2g of biotin, and 5.0ml of sulfuric acid, to which water is added to make a final volume of 1 liter. An example of the composition of K. lactis limited medium (DMSor) is as follows: 11.83 g / L KH2PO4, 2.299 g / L K2HPO4, 20 g / L fermentable sugars (e.g., galactose, maltose, latotriose, sucrose, fructose, or glucose) and / or sugar alcohols (e.g., erythritol, hydrolyzed hydrogenated starch, isomalt, lactitol, maltitol, mannitol, and xylitol), 1 g / L MgSO4.7H2O, 10 g / L (NH4)SO4, 0.3 3 g / L CaCl2.2H2O, 1 g / L NaCl, 1 g / L KCl, 5 mg / L CuSO4.5H2O, 30 mg / L MnSO4.H2O, 10 mg / L ZnCl2, 1 mg / L KI, 2 mg / L CoCl2.6H2O, 8 mg / L Na2MoO4.2H2O, 0.4 mg / L H3BO3, 15 mg / L FeCl3.6H2O, 0.8 mg / L biotin, 20 mg / L calcium pantothenate, 15 mg / L thiamine, 16 mg / L myo-inositol, 10 mg / L nicotinic acid, and 4 mg / L pyridoxine.

[0405] Yeast cells can be cultured in a 48-well deep-well plate sealed with a sterile, breathable cover after seeding. Yeast colonies (e.g., K. lactis cultured on a plate) can be picked up and seeded into a deep-well plate containing 2.2 mL of DMSor medium per well. The seeded deep-well plate can be cultured for 6 days at 23.5°C in a refrigerated incubator shaker while being shaken at 280 rpm. On the 6th day after seeding, the medium is collected by centrifugation at 4000 rpm for 10 minutes and then filtered using a filter plate with a pore size of 0.22 μM. The filtered medium is then subjected to HPLC analysis.

[0406] Examples of yeast strains

[0407] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide is U1 as shown in SEQ ID NO: 1 Compared to the wild-type sequence of -agatoxin-Ta1b, it contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; and X7 is G or absent.

[0408] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. Compared to X1, it contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; and TVP has one amino acid substitution in X1, X2, X3, X4, or X5.

[0409] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises a smaller amount compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. It contains at least one amino acid substitution, where X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, TVP has one amino acid substitution in X1, X2, X3, X4, or X5, where X7 is glycine.

[0410] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of encoding a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide is compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. It contains at least one amino acid substitution, where X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and TVP has one amino acid substitution in X1, X2, X3, X4, or X5, and X7 is absent.

[0411] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises less than the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. Each contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; and TVP has one amino acid substitution in X1, X2, X3, X4, or X5, while X6 and X7 are absent.

[0412] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide is at least 1% identical to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1. It contains one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; and TVP contains the amino sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

[0413] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, and X1 is 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 is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of the following sequence numbers: SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150.

[0414] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is A, S, and X1 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 the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces.

[0415] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises U1-agatoxin-Ta1 as shown in SEQ ID NO: 1. Compared to the wild-type sequence of b, it contains at least one amino acid substitution, where X1 is A, S, or N, X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S, X3 is T or P, X4 is K or A, X5 is R or A, Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R, X6 is K or absent, X7 is G or absent, and the yeast strains are selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

[0416] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (I):EPDEICR-X1-X2-M-X3-NKEFTY-X4-SNVCNNCGDQVAACEAECF-X5-NDVY-Z1-ACHEAQ-X6-X7, and the polypeptide comprises U1-agatoxin-T as shown in SEQ ID NO: 1. Compared to the wild-type sequence a1b, it contains at least one amino acid substitution, where X1 is A, S, or N; X2 is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X3 is T or P; X4 is K or A; X5 is R or A; Z1 is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X6 is K or absent; X7 is G or absent; and the yeast strain is Kluyveromyces lactis or Kluyveromyces marxianus.

[0417] Form (II)

[0418] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q and Z1 is T or A.

[0419] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and if Z1 is T, the TVP is glycosylated.

[0420] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, Z1 is T or A, X1 is Q, and Z1 is A.

[0421] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and the TVP comprises the amino sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 49, or SEQ ID NO: 51.

[0422] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence capable of encoding a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and the TVP is encoded by a polynucleotide sequence or a complementary nucleotide sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 54, or SEQ ID NO: 56.

[0423] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and the yeast strain is selected from any species of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces.

[0424] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and the yeast strain yeast cells are selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

[0425] In some embodiments, the yeast strain of the present invention may comprise a first expression cassette comprising a polynucleotide or a complementary nucleotide sequence thereof capable of functioning to encode a TVP, wherein the TVP comprises an amino acid sequence at least 90% identical to the amino acid sequence of formula (II):EPDEICRA-X1-MTNKEFTYKSNVCNNCGDQVA-ACEAECFRNDVY-Z1-ACHEAQKG, wherein the polypeptide comprises at least one amino acid substitution compared to the wild-type sequence of U1-agatoxin-Ta1b shown in SEQ ID NO: 1, where X1 is R or Q, and Z1 is T or A, and the yeast strain is Kluyveromyces lactis or Kluyveromyces marxianus.

[0426] Yeast transformation, TVP purification, and analysis

[0427] An example of a yeast transformation method is as follows: Yeast cells are transformed with an expression vector containing a TVP-expressing ORF. First, the expression vector is linearized, usually by spe...

Claims

1. An insecticidal U containing the amino sequence shown in any one of the following sequence numbers: SEQ ID NOs: 2-15, 49-53, or 77-110. １ - Agatoxin-Ta1b variant polypeptide (TVP), or an agriculturally acceptable salt thereof.

2. The TVP according to claim 1, wherein the TVP further comprises two or more homopolymers or heteropolymers of TVPs, the amino acid sequences of each TVP being the same or different.

3. The TVP according to claim 1, wherein the TVP is a fusion protein comprising two or more TVPs separated by a cleavable linker or an incleavable linker.

4. The TVP according to claim 3, wherein the cleavable or non-cleavable linker has the amino acid sequence shown in any one of sequence numbers 61 to 70.

5. A composition comprising TVP or an agriculturally acceptable salt thereof and at least one additive, wherein the TVP contains an amino sequence represented by any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

6. The composition according to claim 5, wherein the TVP further comprises two or more homopolymers or heteropolymers of TVP, the amino acid sequences of each TVP being the same or different.

7. The composition according to claim 6, wherein the TVP is a fusion protein comprising two or more TVPs separated by a cleavable linker or an incleavable linker.

8. The TVP according to claim 7, wherein the cleavable or incleavable linker has the amino acid sequence shown in any one of sequence numbers 61 to 70.

9. The one or more additives mentioned above are trehalose, maltodextrin, and anhydrous dipotassium hydrogen phosphate (K ２ HPO ４ ), potassium dihydrogen phosphate (KH ２ PO ４ A composition according to any one of claims 5 to 8, selected from the group consisting of ), BIT, and fermented solids.

10. Based on the total weight of the composition, the TVP is about 2% to about 16% w / w, trehalose is about 5% to about 40% w / w, BIT is about 0.01% to about 0.1% w / w, maltodextrin is about 10% to about 50% w / w, dipotassium hydrogen phosphate anhydrous (K ２ HPO ４ ), is about 1% to about 5% w / w, potassium dihydrogen phosphate (KH ２ PO ４ ), is about 0.10% to about 1% w / w, and the fermented solids are about 15% to about 40% w / w. The composition according to claim 9.

11. The composition comprises approximately 8.5% w / w of TVP, approximately 25% w / w of trehalose, approximately 0.05% w / w of BIT, approximately 36.3% w / w of maltodextrin, and anhydrous dipotassium hydrogen phosphate (K ２ HPO ４ ) is approximately 2.6% w / w, and potassium dihydrogen phosphate (KH ２ PO ４ The composition according to claim 10, wherein the content of ) is approximately 0.4% w / w and the fermented solids content is approximately 26.85% w / w.

12. Insecticide U １ A polynucleotide encoding an agatoxin-Ta1b variant polypeptide (TVP), wherein the TVP contains an amino sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

13. The polynucleotide according to claim 12, wherein the polynucleotide sequence has a nucleotide sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150.

14. A method for generating TVP, wherein the method is (a) Prepare a vector comprising a first expression cassette containing a polynucleotide encoding a TVP that includes an amino sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110. (b) Introducing the vector into a host cell, and (c) Growing the host cells in the growth medium under conditions that enable the expression and secretion of TVP into the growth medium, The method, including the method described above.

15. The method according to claim 14, wherein the vector is a plasmid containing an alpha-MF signal.

16. The method according to claim 15, wherein the TVP is functionally bound to the alpha-MF signal peptide.

17. The method according to claim 16, wherein the alpha-MF signal is functionally capable of expressing the alpha-MF signal peptide.

18. The method according to claim 17, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

19. The method according to claim 18, wherein the eukaryotic cell is a yeast cell.

20. The method according to claim 19, wherein the yeast cells are selected from any species belonging to the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces.

21. The method according to claim 20, wherein the yeast cells are selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

22. A method for controlling, eliminating, or suppressing pests, the method comprising applying a TVP according to any one of claims 1 to 4 or a composition according to any one of claims 5 to 11 in an effective amount as a pesticide to a place where the pests are present or to a plant or animal susceptible to attack by the pests.

23. Acleris gloverana; Acrobasis vaccinii; Aedes aegypti; Agrotis ipsilon; Alsophila pometaria; Amorbia humerosana; Anaphothrips obscrurus; Anarsia lineatella; Anarsia lineatella; Anomala orientalis; Anthonomus eugenii; Anticarsia gemmatalis; Archips argyrospilia; Archips rosanus; Argyrotaenia citrana; Argyrotaenia velutinana; Automeris io; Batrachedra comosae; Busseola fusca; Caudra cautella; Chilo suppressalis; Choristoneura fumiferana; Choristoneura pinus; Choristoneura rosaceana; Colias eurytheme; Cotinis nitida; Crambus sp.; Culex pipiens; Culex quinquefasciatus; Curculio caryae; Curculio occidentis; Cyclocephala borealis; Cyclocephala lurida; Cydia pomonella; Cylas formicarius; Datana integerrima; Datana major; Diabrotica virgifera; Diaphania hyalinata; Diaprepes abbreviatus; Diatraea saccharalis; Dryocampa rubicunda; Ennomos subsignaria; Ennomos subsignaria; Ephestia elutella; Epiphyas postvittana; Erinnyis ello; Erionota thrax; Estigmene acrea; Eumorpha achemon; Evergestis rimosalis; Frankliniella occidentalis; Frankliniella fusca; Frankliniella intonsa; Frankliniella schultzei; Grapholita molesta; Grapholita packardi; Harrisina americana; Helicoverpa armigera; Helicoverpa zea; Heliothis virescens; Hercinothrips femoralis; Heterocampa guttivitta; Homadaula anisocentra; Hypera postica; Hypera zoilus; Hypercompe scribonia; Hyphantria cunea; Hypothenemus hampei; Lambdina fiscellaria; Lasiocampidae sp.; Leptinotarsa ​​decemlineata; Lissorhoptrus oryzophilus; Listronotus maculicollis; Lobesia botrana; Lymantria dispar; Maladera castanea; Manduca quinquemaculata; Manduca sexta; Melissopus latiferreanus; Neohydatothrips variabilis; Neophasia menapia; Noctuid species; Nymphula stagnata; Orgyia pseudotsugata; Orgyia vetusta; Ostrinia furnacalis; Ostrinia nubilalis; Otiorhynchus ovatus; Paleacrita merrickata; Papilio cresphontes; Paralobesia viteana; Pectinophora gossypiella; Peridroma saucia; Pezothrips kellyanus; Phryganidia californica; Phyllophaga sp.; The method according to claim 22, wherein the pest is selected from the group consisting of Pieris rapae, Plathypena scabra, Platynota flavedana, Platynota idaeusalis, Platynota stultana, Platyptilia carduidactyla, Playnota stultana, Plodia interpunctella, Plutella xylostella, Popillia japonica, Pseudaletia unipuncta, Pseudoplusia includens, Rhizotroqus majalis, Sabulodes aegrotata, Schizura concinna, Scirtothrips perseae, Sericothrips variabilis, Sibine stimulea, Southern Diatraea crambidoides, Sphenophorus apicalis, Sphenophorus cariosus, Sphenophorus inaequalis, Sphenophorus minimus, Sphenophorus parvulus, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Syntomeida epilais, Thecla basilides, Thrips palmi, Thrips tabaci, Thymelicus lineola, Thyridopteryx ephemeraeformis, Trichoplusia ni, Xanthogaleruca luteola, and Xylomyges curialis.

24. A vector comprising a polynucleotide encoding a TVP having the amino acid sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

25. The vector according to claim 24, wherein the polynucleotide has a nucleotide sequence shown in any one of SEQ ID NOs: 17-30, SEQ ID NOs: 54-58, or SEQ ID NOs: 117-150.

26. A yeast strain containing TVP that includes the amino sequence shown in any one of the following sequence numbers: SEQ ID NOs: 2-15, 49-53, or 77-110.

27. The yeast strain according to claim 26, wherein the yeast cells are selected from any species belonging to the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, or Schizosaccharomyces.

28. The yeast strain according to claim 27, wherein the yeast cells are selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

29. Insecticide U １ - Agatoxin-Ta1b variant polypeptide (TVP), comprising the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 52, or an agriculturally acceptable salt thereof.

30. Insecticide U １ - Agatoxin-Ta1b variant polypeptide (TVP), wherein the TVP or an agriculturally acceptable salt thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO:

52.

31. Insecticide U １ - Agatoxin-Ta1b variant polypeptide (TVP), comprising the amino acid sequence shown in Sequence ID No. 51, the TVP or an agriculturally acceptable salt thereof.

32. The insecticidal U according to claim 31, wherein the TVP consists of the amino acid sequence shown in Sequence ID No.

51. １ - Agatoxin-Ta1b variant polypeptide (TVP) or an agriculturally acceptable salt thereof.

33. (1) At least one U having the amino acid sequence shown in any one of SEQ ID NOs: 2-15, 49-53, or 77-110 １ - A TVP-insecticidal protein comprising (2) an agatoxin-Ta1b variant polypeptide (TVP) and one or more of the following: (3) an endoplasmic reticulum signal peptide (ERSP); a linker peptide (L); or a translation stabilizing protein (STA).

34. The TVP-insecticidal protein is as follows: TVP-L; (TVP) Ｎ -L; (TVP-L) Ｎ ; L-TVP; L-(TVP) Ｎ ; (L-TVP) Ｎ ; STA-TVP; STA-(TVP) Ｎ ; TVP-STA; (TVP) Ｎ -STA; (STA-TVP) Ｎ ; (TVP-STA) Ｎ ; L-TVP-STA; L-STA-TVP; L-(TVP-STA) Ｎ ; L-(STA-TVP) Ｎ ; L-(TVP) Ｎ -STA; (L-TVP) Ｎ -STA; (L-STA-TVP) Ｎ ; (L-TVP-STA) Ｎ ; (L-STA) Ｎ -TVP; (L-TVP) Ｎ -STA; STA-L-TVP; STA-TVP-L; STA-L-(TVP) Ｎ ; (STA-L) Ｎ -TVP;STA-(L-TVP) Ｎ ; (STA-L-TVP) Ｎ ;STA-(TVP) Ｎ -L;STA-(TVP-L) Ｎ 、;(STA-TVP) Ｎ -L;(STA-TVP-L) Ｎ ; TVP-L-STA;TVP-STA-L;(TVP) Ｎ -STA-L;(TVP-L) Ｎ -STA;(TVP-STA) Ｎ -L;(TVP-L-STA) Ｎ ;(TVP-STA-L) Ｎ ; ERSP-TV;ERP-(TV) Ｎ ;Electronics-TV-L;Electronics-(TV) Ｎ -L;ER@-(TOP-L) Ｎ ;Electricity-Liquidity-TV;Electricity-Liquidity-(TV) Ｎ ; E-TV (L-TV) Ｎ ;Electronics-TV;Electronics-TV-(TV) Ｎ ;Electronics-TV-Story;Electronics-(TV) Ｎ - TV; ESR - (TV) Ｎ ; MRSP-(TV-SA) Ｎ ; ERSP-L-VP-SA;ER-VP-L-VP;ER-L-VP Ｎ ; ERR-L-(ST-TV) Ｎ ; ERSP-L-(TV) Ｎ - TV; E-TV (L-TV) Ｎ - TV; E-TV (L-TV) Ｎ ; MRSP-(L-MEP-SA) Ｎ ; MRSP-(L-SA) Ｎ -TV;ERSP-(L-TV) Ｎ - TV; TV; TV; TV; TV (TV) Ｎ ; MRSP-(SA-L) Ｎ -TV;Electronics-Story-(L-TV) Ｎ ; ERSP-(SA-L-RR) Ｎ ; ERR-ST--(TV) Ｎ -L;ERSP-STA-(TOP-L) Ｎ ; ERSP-(SA-TV) Ｎ -L;ERSP-(STAA-TVP-L) Ｎ ; ERSP-TVP-L-STA; ERSP-TVP-STA-L; ERSP-(TVP) Ｎ -STA-L;ERSP-(TVP-L) Ｎ -STA;ERSP-(TVP-STA) Ｎ -L;ERSP-(TVP-L-STA) Ｎ , or ERSP-(TVP-STA-L) Ｎ The TVP-insecticidal protein according to claim 33, selected from the group consisting of (wherein N is an integer from 1 to 200).

35. The TVP-insecticide protein according to claim 34, wherein the TVP-insecticide protein comprises at least one TVP or two or more TVPs, and the amino acid sequences of each of the two or more TVPs are the same or different.

36. The TVP-insecticide protein according to any one of claims 33 to 35, wherein the linker peptide (L) is a cleavable linker or an incleavable linker.

37. The TVP-insecticide protein according to claim 36, wherein the linker peptide (L) has the amino acid sequence shown in any one of SEQ ID NOs: 31-33 or SEQ ID NOs: 61-70, the ERSP has the amino acid sequence shown in any one of SEQ ID NOs: 37-39, and the STA has the amino acid sequence shown in any one of SEQ ID NOs: 34, 36, or 42.

38. A plant, plant tissue, plant cell, plant seed, or part thereof containing a TVP having an amino sequence shown in any one of SEQ ID NOs: 2-15, SEQ ID NOs: 49-53, or SEQ ID NOs: 77-110.

39. The aforementioned plants include alfalfa, banana, barley, beans, broccoli, cabbage, canola, carrots, cassava, castor beans, cauliflower, celery, chickpeas, Chinese cabbage, citrus fruits, coconuts, coffee, corn, clover, cotton, cucurbits, cucumbers, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, chives, lettuce, loblolly pine, millet, melon, nuts, oats, olives, onions, ornamental plants, palms, pasture grass, peas, peanuts, pepper, pigeon peas, pine, and ginger. The plant, plant tissue, plant cell, plant seed, or plant part thereof according to claim 38, wherein the plant part is a leaf, stem flower, sepal, fruit, root, rhizome, safflower, shrub, sorghum, southern pine, soybean, spinach, pumpkin, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet potato, switchgrass, tea, tobacco, tomato, rye wheat, turfgrass, watermelon, or wheat, and the plant part is a leaf, stem flower, sepal, fruit, root, or seed.

40. A kit comprising (1) one or more TVPs, TVP-insecticide proteins, or agrochemically acceptable salts thereof, and (2) at least one additive, wherein the TVP is selected from any one of claims 1 to 4 or 26 to 32, and the TVP-insecticide protein is selected from any one of claims 33 to 37.

41. The kit according to claim 40, wherein the at least one additive is a carrier, dispersant, wetting agent, antifreeze, thickener, preservative, emulsifier, binder, or adhesive.

42. An insecticidal U containing the amino acid sequence shown in Sequence ID No. 52 １ - Agatoxin-Ta1b variant polypeptide (TVP) or an agriculturally acceptable salt thereof.

43. An insecticidal U consisting of the amino acid sequence shown in Sequence ID No. 52 １ - Agatoxin-Ta1b variant polypeptide (TVP) or an agriculturally acceptable salt thereof.

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