title: Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology.
abstract: 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 Ca(2+) ion concentrations on extracellular recombinant SVP2 expression and stability were investigated. The primary results revealed that the IPTG concentration, Ca(2+) 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 (522 U/ml) was achieved in 0.0089 mM IPTG for 24h at 30 °C, an OD600 of 2, 0.5% of peptone and yeast extract, and a Ca(2+) ion concentration of 1.3 mM. The results exhibited that the minimum level of IPTG concentration along with high cell density and medium level of Ca(2+) with prolonged induction time provided the best culture condition for maximum extracellular production of heterologous protease SVP2 in E. coli expression system.
authors.affiliation: Molecular Biotechnology Lab., Department of Biology, Faculty of Sciences, Shiraz University, P.O. Box 71467-13565 Shiraz, Iran.
authors.forename: Laleh
authors.initials: L
authors.lastname: Beigi
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Karbalaei-Heidari
authors.forename: Mahmood
authors.initials: M
authors.lastname: Kharrati-Kopaei
mesh_terms: D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D045726:Metalloproteases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015233:Models, Statistical; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D012107:Research Design; D014737:Vibrionaceae / Q000201:enzymology / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D045726:Metalloproteases