Insecticidal proteins
A technology for insect resistance and plants, applied in the direction of insecticides, biocides, peptide sources, etc., can solve the problems of increased alternative pest control mechanisms and environmental burdens
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[0236]The present invention may be further described by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [New York] (1984) and Ausubel, F.M. et al., Current Protocols in Molecular Biology, New York [New York ], John Wiley and Sons Inc. [John Wiley and Sons Publishing Company], (1988), Reiter et al., Methods in Arabidopsis Research [Arabidopsis Research Methods], WorldScientific Press [World Science Publishing House] (1992), and Schultz et al., Plant Molecular Biology Manual, K...
example 1
[0237] Example 1: Identification of proteins with insecticidal activity against western corn rootworm
[0238] An insecticidal protein (SEQ ID NO: 1 ) was identified from Woodsholea maritima. An E. coli optimized version of the gene (SEQ ID NO:2) was synthesized and cloned into the pET29a vector, resulting in construct pET29a (Woods). The pET29a (Woods) construct was transformed into E. coli BL21*(DE3) and incubated overnight at 18°C with IPTG in Luria-Bertani medium for protein expression. Soluble fractions of lysates were prepared from these cultures by using a French pressure cell followed by centrifugation of the whole lysate at 20,000 xg for thirty minutes. The supernatant (soluble fraction) was then tested for biological activity against western corn rootworm (WCR).
[0239] Bioactivity assays were performed using the dietary incorporation method. Briefly, E. coli BL21*(DE3) lysates were mixed with an equal volume of heated artificial insect diet (Bioserv, Frenchto...
example 2
[0246] Example 2: Purified WoodsCRW has insecticidal activity against western corn rootworm
[0247] The pET-6His-SUMO construct comprising SEQ ID NO:2 was generated against WoodsCRW. The pET-6His-SUMO-WoodsCRW construct was transformed into E. coli BL21*(DE3) for protein production. The SUMO-tagged protein was purified using standard techniques for His-tagged proteins, followed by cleavage with SUMO protease to release the untagged WoodsCRW protein. The cleaved protein was then applied to a HisTrapFFFPLC column and the column flow-through was collected. The purity of the flow-through fraction was analyzed by SDS-PAGE (expected MW of WoodsCRW 54.1 kDa). Purified WoodsCRW was then dialyzed into IX PBS overnight at 4°C and then concentrated to 13 mg / mL. A range of concentrations of pure protein was then tested against 12 WCR larvae using the dietary incorporation method described in Example 1. As shown in Table 3, WoodsCRW was very effective against WCR; WoodsCRW at 6 μg / m...
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