Overexpression and Characterization of Vibrio mimicus Metalloprotease

  • Shin, Seung-Yeol (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Lee, Jong-Hee (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Huh, Sung-Hoi (Department of Oceanography, Pukyong National University) ;
  • Park, Young-Seo (Department of Food and Bioengineering, Kyungwon University) ;
  • Kim, Jin-Man (Department of Biological Engineering, Yosu National University) ;
  • Kong, In-Soo (Department of Biotechnology and Bioengineering, Pukyong National University)
  • Published : 2000.10.01

Abstract

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.

Keywords

References

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