A genomic library of Bacillus thuringiensis var. kurstaki (B.t.k.) was constructed and it positive clone harboring the full-length gene encoding a novel vegetative insecticidal protein (Vip3V) was characterized. The vip31' 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 917 bp 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 or silkworm (Bombyx mori) and mosquito (Culex quinquefasciatus). (C) 2002 Elsevier Science (USA). All rights reserved.