Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus.
A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the empV gene was expressed in minicells, a unique peptide of approx. 46 kDa was identified. Protease activity staining experiments also indicated a similar M(r) for the protease. The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it. The crude enzyme prepared from the periplasmic fraction of recombinant E. coli was inhibited by a metalloprotease inhibitor and Zn2+ is essential for its protease activity. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long 'pro' sequence consisting of 172 aa. The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V. vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference. The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V. vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV. The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining. The deduced aa sequence of EmpV showed high homology to V. anguillarum metalloprotease (EmpA), V. cholerae HA/protease (HprC), and V proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues.
Chuang
Y C
YC
Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
Chang
T M
TM
Chang
M C
MC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D021382:Protein Sorting Signals / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D008666:Metalloendopeptidases