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Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology

► RSM is a suitable statistical approach for optimization functions. ► SVP2 is a novel thermolysin-like metalloprotease with various industrial applications. ► Salinivibrio proteolyticus is a new isolated moderately halophilic bacterium in the Salinivibrio genus. ► Lower IPTG concentrations together...

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Published in:Protein expression and purification 2012-07, Vol.84 (1), p.161-166
Main Authors: Beigi, Laleh, Karbalaei-Heidari, Hamid Reza, Kharrati-Kopaei, Mahmood
Format: Article
Language:English
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Summary:► RSM is a suitable statistical approach for optimization functions. ► SVP2 is a novel thermolysin-like metalloprotease with various industrial applications. ► Salinivibrio proteolyticus is a new isolated moderately halophilic bacterium in the Salinivibrio genus. ► Lower IPTG concentrations together with longer induction times could be a novel method for high secretion of heterologous proteins in E. coli. In this work, SVP2 from Salinivibrio proteolyticus strain AF-2004, a zinc metalloprotease with suitable biotechnological applications, was cloned for expression at high levels in Escherichia coli with the intention of changing culture conditions to generate a stable extracellular enzyme extract. The complete ORF of SVP2 gene was heterologously expressed in E. coli BL21 (DE3) by using pQE-80L expression vector system. In initial step, the effect of seven factors include: incubation temperature, peptone and yeast extract concentration, cell density (OD600) before induction, inducer (IPTG) concentration, induction time, and Ca2+ ion concentrations on extracellular recombinant SVP2 expression and stability were investigated. The primary results revealed that the IPTG concentration, Ca2+ ion concentration and induction time are the most important effectors on protease secretion by recombinant E. coli BL21. Central composite design experiment in the following showed that the maximum protease activity (522U/ml) was achieved in 0.0089mM IPTG for 24h at 30°C, an OD600 of 2, 0.5% of peptone and yeast extract, and a Ca2+ ion concentration of 1.3mM. The results exhibited that the minimum level of IPTG concentration along with high cell density and medium level of Ca2+ with prolonged induction time provided the best culture condition for maximum extracellular production of heterologous protease SVP2 in E. coli expression system.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2012.05.004