title: Cloning, nucleotide sequence, and expression of Achromobacter protease I gene.
abstract: Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini.
authors.affiliation: Division of Protein Chemistry, Osaka University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ohara
authors.forename: K
authors.initials: K
authors.lastname: Makino
authors.forename: H
authors.initials: H
authors.lastname: Shinagawa
authors.forename: A
authors.initials: A
authors.lastname: Nakata
authors.forename: S
authors.initials: S
authors.lastname: Norioka
authors.forename: F
authors.initials: F
authors.lastname: Sakiyama
mesh_terms: D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase