Proteolytically stable u1-agatoxin-ta1b variant polypeptides for pest control
AssigneeVestaron Corporation
InventorsKyle SCHNEIDER, Alexandra HAASE, Breck DAVIS, Ryan GARRETT
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Abstract
New insecticidal proteins, nucleotides, peptides, and their expression in plants. Methods of producing new nucleotides and new peptides, new processes, new production techniques, new formulations, and new organisms. The present disclosure is also related to and describes novel peptides called TVPs, which are non-natural peptide variations based upon the U1-agatoxin-Ta1b toxin derived from the Hobo spider. Here, we describe: genes encoding TVPs; formulations and combinations comprising TVP genes and/or peptides; and methods using the same that are useful for the control of pests.
Claims148
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (I):
- E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1 b as set forth in SEQ ID NO:1, and 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;
- or a pharmaceutically acceptable salt thereof.
A composition consisting of a TVP, and one or more excipients;
- wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (I) E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and 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;
- or a pharmaceutically acceptable salt thereof;
- and wherein the one or more excipients is selected from the group consisting of:
- trehalose;
- maltodextrin;
- maltose;
- potassium phosphate dibasic (K2HPO4);
- potassium phosphate monobasic (KH2PO4);
- lignosulfonate;
- gypsum;
- sorbitol;
- sodium benzoate;
- potassium sorbate;
- EDTA;
- benzisothiazolinone (BIT);
- and fermentation solids.
A composition consisting of a TVP, and a plurality of excipients;
- wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (I):
- E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and 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;
- or a pharmaceutically acceptable salt thereof;
- wherein the composition consists of an amount of TVP that is 8.5% w/w of the total weight of the composition;
- and wherein the plurality of excipients consists of the following:
- an amount of trehalose that is 25% w/w;
- an amount of BIT that is 0.05% w/w;
- an amount of maltodextrin that is 36.3% w/w;
- an amount of potassium phosphate dibasic anhydrous (K2HPO4) that is 2.6% w/w;
- an amount of potassium phosphate monobasic (KH2PO4) that is 0.4% w/w;
- and an amount of fermentation solids that is 26.85% w/w, of the total weight of the composition.
A polynucleotide encoding a TVP, said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (I):
- E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the TVP comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1 b as set forth in SEQ ID NO:1, and 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;
- or a complementary nucleotide sequence thereof.
The polynucleotide of any one of claims 46-53, wherein if Z1 is T or S, then the TVP is glycosylated.
A method of producing a TVP, the method comprising:
- (a) preparing a vector comprising a first expression cassette comprising a polynucleotide operable to encode a TVP, or complementary nucleotide sequence thereof, said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (I):
- E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and 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 a host cell;
- and
- (c) growing the host cell in a growth medium under conditions operable to enable expression of the TVP and secretion into the growth medium.
A method of protecting a plant from insects comprising, providing a plant which expresses a TVP, or polynucleotide encoding the same.
A vector comprising a polynucleotide operable to encode a TVP having an amino acid sequence with 90% similarity to a sequence as set forth in any one of SEQ ID NOs:
- 2-15, 49-53, or 77-110.
A yeast strain comprising:
- a first expression cassette comprising a polynucleotide operable to encode a TVP, said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (I):
- E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and 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;
- or complementary nucleotide sequence thereof.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (II):
- E-P-D-E-I-C-R-A-X1-M-T-N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-R-N-D-V-Y-Z1-A-C-H-E-A-Q-K-G;
- wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and wherein X1 is R or Q;
- and Z1 is T or A;
- or a pharmaceutically acceptable salt thereof.
A composition consisting of a TVP, and one or more excipients;
- wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (II):
- E-P-D-E-I-C-R-A-X1-M-T-N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-R-N-D-V-Y-Z1-A-C-H-E-A-Q-K-G;
- wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1 b as set forth in SEQ ID NO:1, and wherein X1 is R or Q;
- and Z1 is T or A;
- or a pharmaceutically acceptable salt thereof;
- and wherein the one or more excipients is selected from the group consisting of:
- trehalose;
- maltodextrin;
- maltose;
- potassium phosphate dibasic (K2HPO4);
- potassium phosphate monobasic (KH2PO4);
- lignosulfonate;
- gypsum;
- sorbitol;
- sodium benzoate;
- potassium sorbate;
- EDTA;
- benzisothiazolinone (BIT);
- and fermentation solids.
A composition consisting of a TVP, and a plurality of excipients;
- wherein the TVP comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to Formula (II):
- E-P-D-E-I-C-R-A-X1-M-T-N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-R-N-D-V-Y-Z1-A-C-H-E-A-Q-K-G;
- wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of U1-agatoxin-Ta1b as set forth in SEQ ID NO:1, and wherein X1 is R or Q;
- and Z1 is T or A;
- or a pharmaceutically acceptable salt thereof;
- wherein the composition consists of an amount of TVP that is 8.5% w/w of the total weight of the composition;
- and wherein the plurality of excipients consists of the following:
- an amount of trehalose that is 25% w/w;
- an amount of BIT that is 0.05% w/w;
- an amount of maltodextrin that is 36.3% w/w;
- an amount of potassium phosphate dibasic anhydrous (K2HPO4) that is 2.6% w/w;
- an amount of potassium phosphate monobasic (KH2PO4) that is 0.4% w/w;
- and an amount of fermentation solids that is 26.85% w/w, of the total weight of the composition.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs:
- 2, 49, or 51;
- or a pharmaceutically acceptable salt thereof.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP comprising an amino acid sequence set forth in any one of SEQ ID NOs:
- 2, 49, or 51;
- or a pharmaceutically acceptable salt thereof.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP consisting of an amino acid sequence set forth in any one of SEQ ID NOs:
- 2, 49, or 51;
- or a pharmaceutically acceptable salt thereof.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO 51, or a pharmaceutically acceptable salt thereof.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP comprising an amino acid sequence set forth in SEQ ID NO:
- 51, or a pharmaceutically acceptable salt thereof.
An insecticidal U1-agatoxin-Ta1b variant polypeptide (TVP), said TVP consisting of an amino acid sequence set forth in SEQ ID NO:
- 51, or a pharmaceutically acceptable salt thereof.
Dependencies are inferred from the claim text.