title: Molecular cloning and expression of proteinaceous alpha-amylase inhibitor gene from Streptomyces nitrosporeus in Escherichia coli.
abstract: The proteinaceous alpha-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous alpha-amylase inhibitors.
authors.affiliation: Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Sumitani
authors.forename: T
authors.initials: T
authors.lastname: Kawaguchi
authors.forename: N
authors.initials: N
authors.lastname: Hattori
authors.forename: S
authors.initials: S
authors.lastname: Murao
authors.forename: M
authors.initials: M
authors.lastname: Arai
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C083183:T-76 protein, Streptomyces nitrosporeus; D000516:alpha-Amylases