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Expression in Escherichia coli of the recombinant Vibrio anguillarum metalloprotease and its purification and characterization

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The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca2+, Na+ and Mg2+ had an activating effect on the enzyme while Zn2+ and Cu2+ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis.

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This research was supported by grants 3032802 from National Natural Science Foundation of China and LuKaRenCaiGongCheng program from Ocean University of China.

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Correspondence to Z. M. Chi.

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Zhang, F.L., Chi, Z.M., Zhu, K.L. et al. Expression in Escherichia coli of the recombinant Vibrio anguillarum metalloprotease and its purification and characterization. World J Microbiol Biotechnol 23, 331–337 (2007). https://doi.org/10.1007/s11274-006-9228-z

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  • empA gene
  • Vibrio anguillarum
  • recombinant metalloprotease
  • expression
  • characterization