Characterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C-terminal deletions.
To investigate the enzymatic properties of Vibrio mimicus metalloprotease, the mature metalloprotease gene (vmc) was overexpressed in Escherichia coli and the recombinant protein (rVMC61) was purified by metal affinity chromatography. rVMC61 showed maximum activity at about 37 degrees C, pH 8. The purified rVMC61 was very specific toward collagen substrates, such as gelatin, type I, II, and III collagens and synthetic peptides (Cbz-GPLGP and Cbz-GPGGPA). But it did not show degrading activity toward other biological proteins including lysozyme, lactoferrin and bovine serum albumin. rVMC61 also showed cytotoxicity against CHSE-214 fish cells. To examine the role of the C-terminal region of rVMC61, the 3' end of the metalloprotease gene (vmc) was digested serially with exonuclease III. The truncated vmc derivatives encoding 57-42 kDa of the protease were isolated and overexpressed in E. coli. The collagenase activities of truncated proteins were investigated using gelatin as substrate. Deletion of 100 amino acids from the C-terminus resulted in loss of gelatin degrading activity. However, deletion of 67 amino acids from the C-terminus did not affect its gelatin degrading activity.
Lee
Jong-Hee
JH
Department of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, South Korea.
Ahn
Sun-Hee
SH
Lee
Eun-Mi
EM
Kim
Young-Ok
YO
Lee
Sang-Jun
SJ
Kong
In-Soo
IS
D000818:Animals; D002460:Cell Line; D004625:Embryo, Nonmammalian / Q000166:cytology; D004789:Enzyme Activation / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D017434:Protein Structure, Tertiary; D012474:Salmon; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics*
D008666:Metalloendopeptidases