精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。

Secretory expression of arginine deiminase in E. coli

The arginine deiminase （ADI） encoding gene arcA of P. plecoglossicida CGMCC2039 was cloned into secreted expression vector pBAD/gⅢ B under araBAD promoter （pBAD）, and the resulted recombinant plasmid pBAD-ADI was transformed into E. coli TOP10F＇. The effect of induction conditions on ADI expression was investigated, including L-arabinose concentration, induction temperature, and induction time. The optimum induction conditions were determined to be 0. 002 % L-arabinose, 25 ℃ and 5 h, and the ADI activity of whole cells was 68 mU/mL broth. The recombinant ADI in periplasmic space was released by osmotic shock, the ADI activity in periplasmic and cellular fraction were 53 mU/mL broth and 34 mU/mL broth, respectively. SDS-PAGE analysis showed that the molecular mass of recombinant ADI was about 46 kD.