# US 10,117,433 B2 · Pesticides

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- Number: US 10,117,433 B2
- Document ID: US10117433B2
- Status: Granted
- Kind: B2
- Application number: 14/008,412
- Filed: 2012-03-29
- Published: 2014-12-11
- Granted: 2018-11-06
- Earliest claimed: 2011-03-31
- Assignees: University of Durham; The Secretary of State for Environment, Food and Rural Affairs
- Applicants: John A. Gatehouse; Elaine C. Fitches
- Inventors: John A. Gatehouse; Elaine C. Fitches
- Web page: https://patentgrep.com/patent/US10117433B2

## CPC

- A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46)
- A01N 37/46: N-acyl derivatives
- A01N 43/50: 1,3-Diazoles; Hydrogenated 1,3-diazoles
- C07K 14/42: Lectins, e.g. concanavalin, phytohaemagglutinin
- C07K 14/43518: {from spiders}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/8279: {for biotic stress resistance, pathogen resistance, disease resistance}
- C12N 15/8285: {for nematode resistance}
- C12N 15/8286: {for insect resistance}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/21: containing a His-tag
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to the development of fusion proteins based on peptide toxins as pesticides. The invention also relates to transgenic plants encoding said peptides and their utility in pest management.

## Claims

### Claim 1

A fusion protein comprising: (i) an omega atracotoxin family member 1a protein from Hadronyche versuta (ω-ACTX-Hv1a) protein toxin comprising the amino acid sequence: SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD (SEQ ID NO: 1), or a variant of ω-ACTX-Hv1a protein toxin having at least 95% identity to SEQ ID NO: 1, operably linked to (ii) a protein capable of mediating translocation of the fusion protein from the invertebrate gut, wherein the protein capable of mediating translocation is a snowdrop lectin (GNA) comprising the sequence of SEQ ID NO: 3 and wherein the fusion protein comprises the amino acid sequence of any of SEQ ID NOs: 4, 5, 6, 7, 8 or 9.

### Claim 2 (depends on 1)

The fusion protein of claim 1 wherein the fusion protein further comprises an affinity tag to aid purification.

### Claim 3 (depends on 1)

The fusion protein of claim 1 wherein the protein has pesticide activity against invertebrate pests selected from the group consisting of insects, arthropods, nematodes, and molluscs.

### Claim 4 (depends on 1)

A method of preparing a fusion protein comprising culturing a host cell comprising a nucleic acid encoding the fusion protein of claim 1 under conditions suitable for expression of the fusion protein.

### Claim 5 (depends on 4)

The method of claim 4 further comprising the step of recovering the fusion protein.

### Claim 6 (depends on 1)

A pesticide composition comprising the fusion protein of claim 1 in an effective pesticidal amount, together with one or more suitable carriers, diluents, adjuvants, preservatives, dispersants, solvents, and emulsifying agents.

### Claim 7 (depends on 1)

A method for the preparation of a pesticide composition which comprises mixing an effective pesticidal amount of the fusion protein of claim 1 with one or more suitable carriers, diluents, adjuvants, preservatives, dispersants, solvents, or emulsifying agents.

### Claim 8 (depends on 1)

A method of preventing or treating a pest infection of a plant comprising applying a quantity of a composition comprising the fusion protein according to claim 1 to the plant or its locus of growth.

### Claim 9 (depends on 1)

A method of preventing or treating a mollusc or nematode pest infection of a plant comprising applying a quantity of a composition comprising the fusion protein according to claim 1 to the plant or its locus of growth; or introducing to the plant a nucleic acid sequence encoding said fusion protein.

### Claim 10 (depends on 9)

The method of claim 9 wherein the pest is a slug or snail.

### Claim 11 (depends on 1)

A molluscicide bait composition comprising the fusion protein of claim 1.

### Claim 12 (depends on 1)

A transgenic plant or progeny thereof comprising the fusion protein of claim 1.

### Claim 13 (depends on 6)

A method of preventing or treating a pest infection of a plant comprising applying a quantity of the pesticide composition according to claim 6, to the plant or its locus of growth.

### Claim 14 (depends on 6)

A method of preventing or treating a mollusc or nematode pest infection of a plant comprising applying a quantity of the pesticide composition according to claim 6, to the plant or its locus of growth; or introducing to the plant a nucleic acid sequence encoding said fusion protein by transformation with Agrobacterium, particle bombardment, electroporation or viral transformation.

### Claim 15 (depends on 6)

A molluscicide bait composition comprising the pesticide composition according to claim 6.

### Claim 16 (depends on 1)

The fusion protein of claim 1 wherein the fusion protein comprises a w-ACTX-Hv1a protein toxin having the sequence of SEQ ID NO: 1.

### Claim 17 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein comprises a variant of ω-ACTX-Hv1 a protein toxin having at least 95% identity to SEQ ID NO: 1, and wherein amino acid K34 of the ω-ACTX-Hv1a protein toxin of SEQ ID NO: 1 is modified.

### Claim 18 (depends on 17)

The fusion protein of claim 17 wherein the variant of ω-ACTX-Hv1a protein toxin has a K34Q substitution.