Cloning and expression of the vegetative insecticidal protein (vip3V) gene of Bacillus thuringiensis in Escherichia coli.
A genomic library of Bacillus thuringiensis var. kurstaki (B.t.k.) was constructed and a positive clone harboring the full-length gene encoding a novel vegetative insecticidal protein (Vip3V) was characterized. The vip3V gene was subcloned into pET-22b(+) vector and overexpressed in Escherichia coli to an extent of about 30% of the total protein. While transcription was influenced by the T7 promoter of the vector, synthesis of Vip3V in E. coli host occurred from the B.t.k. ribosomal binding site (rbs) found 917bp downstream of the insert and not from the E. coli rbs of the vector. The expressed Vip3V protein was found in the soluble and periplasmic fractions as well as in the inclusion bodies. A simplified anion-exchange chromatographic method for the purification of Vip3V using step gradient or one-step elution was developed. The purified protein showed broad-spectrum activity against some of the lepidopteran larvae tested and did not show any activity against the larvae of silkworm (Bombyx mori) and mosquito (Culex quinquefasciatus).
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D007306:Insecticides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007915:Lepidoptera / Q000187:drug effects; D008969:Molecular Sequence Data; D010572:Pest Control, Biological; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D007306:Insecticides; C447139:Vip3A protein, Bacillus thuringiensis