Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
New insecticidal proteins, nucleotides, peptides, their expression in plants, methods of producing the peptides, new processes, production techniques, new peptides, new formulations, and new organisms, a process which increases the insecticidal peptide production yield from yeast expression systems. The present invention is also related and discloses selected endotoxins we call cysteine rich insecticidal peptides (CRIPS) which are peptides derived from Bacillus thuringiensis (Bt) and their genes and endotoxins in combination with toxic peptides known as Inhibitor Cystine Knot (ICK) genes and peptides as well as with other types of insecticidal peptides such as trypsin modulating oostatic factor (TMOF) peptide sequences used in various formulations and combinations; of both genes and peptides, useful for the control of insects.
1. A polypeptide comprising a plant Endoplasmic Reticulum Signal Peptide (ERSP) operably linked to one or more ICK insecticidal venom peptides (“ICK insecticidal peptides”), wherein the ICK insecticidal peptides are one or more of: a U-ACTX polypeptide, a Kappa-ACTX polypeptide, an Omega-ACTX polypeptide, or combinations thereof, and wherein said plant ERSP is the N-terminal of said polypeptide.
2. The polypeptide of claim 1 wherein said plant ERSP is a peptide of 5 to 50 amino acids in length.
3. The polypeptide of claim 1 wherein the plant ERSP is linked to from about 2 to 100 ICK insecticidal peptides.
4. The polypeptide of claim 3 wherein the ICK insecticidal peptides may be linked to and separated from each other by one or more or both of the following: 1) a Translational Stabilizing Protein (STA), and wherein said STA may be the same peptide as the ICK insecticidal peptide, or it can be a different peptide or 2) an Intervening Linker Peptide (LINKER) or the ICK insecticidal peptides may be separated from each other by both a STA and a LINKER, in any combination.
5. The polypeptide of claim 4 wherein the ICK insecticidal peptides are separated from each other by one or more Translational Stabilizing Proteins (STAs).
6. The polypeptide of claim 4 wherein the ICK insecticidal peptides are separated from each other by one or more Intervening Linker Peptides (LINKERs).
7. The polypeptide of claim 6 wherein the ICK insecticidal polypeptides are all the same peptide and they are separated from each other by one or more Intervening Linker Peptides (LINKERs).
8. The polypeptide of claim 7 wherein said ICK insecticidal peptide is an U-ACTX insecticidal peptide or an Omega-ACTX insecticidal peptide and the plant ERSP is on the N-terminal of said ICK insecticidal peptide and a Translational Stabilizing Protein (STA) or a intervening linker peptide (LINKERs) may be on either the N-terminal side of the ICK insecticidal peptide, and between the ERSP and the ICK insecticidal peptide or said STA or LINKER may be on the C-terminal side of the ICK insecticidal peptide and said ICK insecticidal peptide is on the C-terminal side of said plant ERSP.
9. The polypeptide of claim 1 wherein said ICK insecticidal peptide is a U-ACTX insecticidal peptide.
10. The polypeptide of claim 8 wherein said ICK insecticidal peptide is fused to a LINKER.
11. The polypeptide of claim 10 wherein said ICK insecticidal peptide is an U-ACTX polypeptide.
12. The polypeptide of claim 11 wherein the ICK insecticidal peptide is selected from native U-ACTX-Hv1a, U+2 U-ACTX-Hv1a, or other forms of U-ACTX-Hv1a.
13. The polypeptide of claim 12 wherein the plant ERSP is selected from a BAAS signal peptide (BGIH), an Extensin signal peptide (EGIH) and a modified Extensin signal peptide (E*GIH).
14. The polypeptide of claim 9 comprising both a LINKER and a STA, said polypeptide having a designation from N-terminus to C-terminus: ERSP-STA-(LINKER 1 -ICK) N or ERSP-(ICK-LINKER 1 -) N -STA; and wherein the subscript N indicates a number of repeats ranging from 1 to 200.
15. The polypeptide of claim 14 , wherein said STA, LINKER and ICK are oriented from N-terminus to C-terminus as: ERSP-STA-LINKER-ICK; ERSP-ICK-LINKER-STA; ERSP-STA-ICK-LINKER; ERSP-ICK-STA-LINKER; ERSP-LINKER-STA-ICK; or ERSP-LINKER-ICK-STA.
16. The polypeptide of claim 4 , wherein the polypeptide comprises a structure from N-terminus to C-terminus as: ERSP-STA-ICK or ERSP—ICK-STA.
17. The polypeptide of claim 4 , wherein the polypeptide comprises a structure from N-terminus to C-terminus as: ERSP-LINKER-ICK-(LINKER-ICK) N or ERSP-ICK-LINKER-(ICK-LINKER) N wherein the integer N indicates a number ranging from 1 to 200.
18. A method for controlling insects wherein a polypeptide of claim 1 in solid or liquid form is provided to either the insect, the locus of the insect or as a plant incorporated protectant.