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Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides

A cysteine, cystine knot technology for use in the fields of toxic peptide production, peptide expression in plants and combination of cysteine-rich peptides

Inactive Publication Date: 2015-03-11
VESTARON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, most insecticidal peptides are sufficiently fragile or not toxic enough to be commercially available
Second, insecticidal peptides are difficult and expensive to produce commercially
Third, many insecticidal peptides degrade rapidly and have short half-lives
Fifth, most of the identified insecticidal peptides have their systemic distribution in insects blocked and / or lose their toxic properties when ingested by insects

Method used

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  • Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
  • Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
  • Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0244] Expression comparison between two transient plant expression systems .

[0245] Transient plant transformation technology was used to rapidly optimize ICK motif protein expression ORF for plant expression. The technique of Agrobacterium infection with plant viral vectors was used here for transient plant transformation due to its high efficiency, simplicity and cheapness. Here, two viral transient plant expression systems are evaluated for ICK motif protein expression in plants. One is the tobacco mosaic virus overexpression system (TRBO, Lindbo JA, Plant Physiology, 2007, V145: 1232-1240). The TRBO DNA vector has a T-DNA region for the Agrobacterium infection method, which contains the CaMV 35S promoter driving the expression of tobacco mosaic virus RNA without the gene encoding the viral coat protein. Another viral transient plant expression system is the FECT expression system (Liu Z and Kearney CM, BMC Biotechnology, 2010, 10:88). The FECT vector also contains ...

Embodiment 2

[0266] Transient expression of ICK motif proteins in tobacco leaves is accompanied by accumulation in distinct subcellular targets .

[0267] Plant-expressed ICK motif proteins need to accumulate to a certain level in plants to effectively protect plants from pests. The accumulation level of plant-expressed ICK-motif proteins can be influenced by their ultimate localization in plant cells. In this example, we investigated the effect of different subcellular localizations of plant expressed ICK motif proteins on protein accumulation levels in plants (using the FECT transient plant expression system). In this example, three subcellular targets were studied, the plant cell wall apoplast (APO), endoplasmic reticulum (ER) and cytoplasm (CYTO).

[0268] The APO-directed ICK motif protein expression ORF was designed to encode a series of translationally fused structural motifs, which can be described as follows: N'-ERSP-Sta-L-ICK-C'. The ICK motif protein in this study was U-ACTX...

Embodiment 3

[0283] Alternative signal peptides for expression of ICK motif proteins in plants .

[0284] Since ER signal peptides can function at the protein expression level, two other ERSPs were tested using the FECT expression system described in the previous examples. Two ERSP candidates are the tobacco extensin signal peptide, abbreviated "E" in this study (Memelink et al., the Plant Journal, 1993, V4: 1011-1022), and one of its variants is abbreviated "E*" (Pogue GP et al., Plant Biotechnology Journal, 2010, V8: 638-654). Their amino acid sequences are listed below (N' to C', one-letter code, non-identical residues are indicated in bold):

[0285] Extensin signal peptide (EMGKMASLFA SL LVVLVSLSLASESSA (SEQ ID NO: 18)

[0286] Extensin signal peptide variant (E*): MGKMASLFA TF LVVLVSLSLASESSA (SEQ ID NO: 19)

[0287] The DNA sequence encoding E was designed for tobacco expression as follows:

[0288] ATGGGTAAGATGGCTTCTCTGTTTGCTTCTCTGCTGGTTGTTCTGGTTCTCTGTCTCTGGCTTCTGAATCTTCT...

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Abstract

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.

Description

[0001] References to related applications [0002] This application is a PCT application claiming earlier U.S. Provisional Application Serial No. 61 / 608,921 filed March 9, 2012, U.S. Provisional Application Serial No. 61 / 644,212 filed May 8, 2012, September 7, 2012 The benefit of U.S. Provisional Application Serial No. 61 / 698,261 filed and U.S. Provisional Application Serial No. 61 / 729,905, filed November 26, 2012, the entire contents of which are incorporated herein by reference. field of invention [0003] Describes and claims new pesticidal proteins, nucleotides, peptides, their expression in plants, methods of producing said peptides, new treatments, production techniques, new peptides, new formulations and production ratios for Combinations of new and known organisms for higher yields expected of related peptides for insect control. Background of the invention [0004] The global safety of food produced through modern agriculture and horticulture is challenged b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/82A01N63/02
CPCA01N63/02C07K14/43518C07K14/43504C07K2319/55C12N15/8286A01N63/10A61P33/14C07K14/325A01N63/50Y02A40/146A01N63/23A01N63/16C07K2319/04C12N2511/00A01P7/04C12N15/8205
Inventor R.M.肯尼迪W.特德福德C.亨德里克森R.维纳布尔C.富涅J.麦金泰尔A.卡尔森鲍林
Owner VESTARON
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