CLONING AND CHARACTERIZATION OF THE GENE (EMP-V) ENCODING EXTRACELLULAR METALLOPROTEASE FROM VIBRIO-VULNIFICUS

A gene (empV) encoding the extracellular metalloprotease of Vibrio vul nificus CKM-1 has been cloned and sequenced. When the empV gene was ex pressed in minicells, a unique peptide of approx. 46 kDa was identifie d. Protease activity staining experiments also indicated a similar M-r for the protease. The empV gene product (EmpV) is secreted into the p eriplasm of Escherichia coli, but not out of it. The crude enzyme prep ared from the periplasmic fraction of recombinant E. coli was inhibite d by a metalloprotease inhibitor and Zn2+ is essential for its proteas e activity. Nucleotide sequence analysis predicted a single open readi ng frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long 'pro' sequenc e consisting of 172 aa. The N-terminal 20 aa sequence for the elastoly tic protease (EepV), purified from the culture supernatant of V. vulni ficus 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 ma ture protein were 5.86 and 44.3 kDa, respectively, which are nearly id entical to those of V. vulnificus L-180 extracellular neutral metallop rotease (EnmV) and of strain ATCC 29307 EepV. The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of th e 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 proteas e (NprP), particularly with respect to active-site residues, zinc-bind ing residues, and cysteine residues. (C) 1997 Elsevier Science B.V.