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.
Park
Hyeon Ung
HU
Department of Microbiology, College of Natural Sciences and Research Center for Molecular Microbiology, Seoul National UniversitySeoul 151-742Korea.
Lee
Kye Joon
KJ
Department of Microbiology, College of Natural Sciences and Research Center for Molecular Microbiology, Seoul National UniversitySeoul 151-742Korea.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development; D014162:Transfection; D065093:beta-Lactamase Inhibitors
D001426:Bacterial Proteins; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; D005947:Glucose