Cloning,Expression and Purification of Arginine Deiminase from Pseudomonas plecoglossicida CGMCC2039 in Escherichia coli BL21(DE3)

Abstract:

Arginine deiminase(ADI),an arginine degrading enzyme,has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors,such as melanomas and hepatocellular carcinomas(HCCs).In this study,the arcA gene that encodes ADI was cloned from P.plecoglossicida CGMCC2039 which was isolated and identified by our laboratory previously.The arcA gene was subcloned into expression vector pET24a and its product ADI was over-expressed in Escherichia coli BL21(DE3).The effects of different IPTG concentrations,induction temperatures and induction time were investigated.Under the optimal expression conditions the enzyme activity reached to 2.04 U/mL broth.The rADI was purified using DEAE Fast Flow anion exchange and SuperdexTM 200 gel filtration column chromatography.The rADI had a molecular mass of about 92.6 kDa,and comprised a homodimer of 46 kDa in the native condition.The specific activity of rADI was determined to be 20.9 U/mg at pH 6.0 and 37 ℃.