Open AccessCloning and heterologous expression of the gene for BLIP-II, a -lactamase-inhibitory protein from Streptomyces exfoliatus SMF19
Author(s) -
Hyeon Ung Park,
Kye Joon Lee
Publication year - 1998
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.019
H-Index - 179
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-144-8-2161
Subject(s) - biology , cosmid , nucleic acid sequence , microbiology and biotechnology , gene , peptide sequence , escherichia coli , amino acid , heterologous expression , streptomyces , recombinant dna , signal peptide , molecular cloning , biochemistry , genetics , bacteria
A -lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF19 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 -lactamase-inhibitory activity was recovered after refolding. In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the me /C1 promoter. The BLIP-II protein produced in recombinant strains of S. lividans was secreted into the culture supernatant in a biologically active form.