Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.
The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.
Lampel
J S
JS
Department of Microbiology, Ohio State University, Columbus 43210.
Aphale
J S
JS
Lampel
K A
KA
Strohl
W R
WR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D010450:Endopeptidases / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D008666:Metalloendopeptidases; D008894:Milk Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D015032:Zinc / Q000494:pharmacology
D008894:Milk Proteins; D011480:Protease Inhibitors; D003035:Cobalt; D010450:Endopeptidases; D008666:Metalloendopeptidases; C074159:neutral proteinase snpA, Streptomyces; D015032:Zinc