Cloning and heterologous expression of the gene for BLIP-II, a beta-lactamase-inhibitory protein from Streptomyces exfoliatus SMF19.

A beta-lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF 19 and its N-terminal amino acid sequence was determined. A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II. The bliB gene was isolated and sequenced. Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence. bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but beta-lactamase-inhibitory activity was recovered after refolding. In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the melC1 promoter. The BLIP-II protein produced in recombinant strains of S. lividans was secreted into the culture supernatant in a biologically active form.