Molecular cloning and expression of proteinaceous alpha-amylase inhibitor gene from Streptomyces nitrosporeus in Escherichia coli.
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.
Sumitani
J
J
Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Japan.
Kawaguchi
T
T
Hattori
N
N
Murao
S
S
Arai
M
M
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*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C083183:T-76 protein, Streptomyces nitrosporeus; D000516:alpha-Amylases