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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Austin B, Austin DA (1999) Bacterial fish pathogens: diseases in farmed and wild fish, 3rd edn. Springer, United Kingdom pp 2–30, ISBN 1852331208
Chen JX, Li Y, Wang XH, Du ZJ, Yu DH, Ji WS, Xu HS (2002a) Cloning and sequencing of metalloprotease gene of a pathogenic Vibrio anguillarum. High Technol Lett 6:106–110
Chen JX, Li Y, Wang XH, Du ZJ, Yu DH, Ji WS (2002b) Purification of an extracellular protease from Vibrio anguillarum and its physicochemical properties. J Fish Sci China 9:318–322
Chi Z, Liu ZQ, Ma CL, Gao, LM (2006) Marine yeasts and their applications in mariculture. J Ocean Univ China, in press
Denkin SM, Nelson DR 2004 Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence. Appl Environ Microbiol 70:4193–4204
Fukasawa S, Nakamura K, Miyahira M, Kurata M (1988) Some properties of two proteinases from luminous bacteria, Vibrio harveyi strain FLN-108. J Biol Chem 52:3009–3014
Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Harlow E, Lane D (1988) Antibodies: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York: pp.20–40, ISBN 0–87969–314–2
Inamura H, Nakai T, Muroga K (1985) An extracellular protease produced by Vibrio anguillarum. Bull Japan Soc Sci Fish 51:1915–1920
Kurien BT, Scofield RH (2003) Protein blotting: a review. J␣Immunol Meth 274:1–15
Lee JH, Ahn SH, Lee EM, Kim YO, Lee DJ, Kong IS (2003) Characterization of the enzyme activity of an extracellular metalloprotease (VMC) from Vibrio mimicus and its C-terminal deletions. FEMS Microbiol Lett 223:293–300
Lee JH, Kim GT, Lee JY, Jun HK, Yu JH, Kong IS (1998) Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus. Biochim Biophys Acta 1384:1–6
Liu PC, Lee KK, Tu CC, Chen SN (1997) Purification and characterization of a cysteine protease produced by pathogenic luminous Vibrio harveyi. Curr Microbiol 35:32–39
Martone CB, Spivak E, Busconi L, Folco EJE, Sanchez JJA (1999) Cysteine protease from myxosporean degrades host myofibrils in vitro. Comparative Biochem Physiol Part B 123:267–272
Miyoshi S, Kawata K, Tomochika K, Shinoda S, Yamamoto S (2001) The C-terminal domain promotes the hemorrhagic damage caused by Vibrio vulnificus metalloprotease. Toxicology 39:1883–1886
Miyoshi S, Sonoda Y, Wakiyama H, Rahman MM, Tomochika K, Shinoda S, Yamamoto S, Tobe K (2002) An exocellular thermolysin-like metalloprotease produced by Vibrio fluuvialis: purification, characterization, and gene cloning. Microb Pathol 33:127–134
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, New York: pp. 260–280, ISBN 0–87969–309–6
Staroscik AM, Denkin SM, Nelson DR (2005) Regulation of the Vibrio anguillarum metalloprotease EmpA by posttranslational modification. J Bacteriol 187:2257–2260
Teo JWP, Zhang LH, Poh CL (2003) Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6. Gene 303:147–156
Zhang FL, Chen JX, Chi Z, Wu LF (2006) Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli. Arch Microbiol, in press
This research was supported by grants 3032802 from National Natural Science Foundation of China and LuKaRenCaiGongCheng program from Ocean University of China.
About this article
Cite this article
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
- empA gene
- Vibrio anguillarum
- recombinant metalloprotease