精氨酸脱亚胺酶基因在大肠杆菌中的克隆、表达及纯化

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

摘要:

通过PCR扩增得到菌株编码ADI的arcA基因,并构建表达载体pET24a-ADI。将该载体导入大肠杆菌BL21(DE3),获得高效表达ADI基因的重组菌。对ADI诱导表达条件进行优化,结果发现宿主菌在A600达到1.0时加入0.2 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG),在30℃诱导4 h酶活最高,为2.04 U/mL发酵液。经超声波破碎、HiPrep DEAE FF阴离子交换层析、SuperdexTM200凝胶过滤层析,可获得SDS-PAGE电泳纯重组精氨酸脱亚胺酶(rADI)。rADI相对分子质量大小为92 600,由两个相同亚基组成,纯化后的rADI比酶活达20.9 U/mg。

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 ℃.