精氨酸脱亚胺酶的克隆表达和纯化  

Cloning,expression and purification of arginine deiminase from Mycoplasma hominis

作　　者：魏星[1] 苏勇[1] 张加慧 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]杭州北斗生物技术有限公司,杭州310011

出　　处：《世界临床药物》2013年第5期280-284,共5页World Clinical Drug

摘　　要：目的研究人型支原体来源的精氨酸脱亚胺酶(MhADI)在大肠杆菌中的表达及纯化。方法人工合成密码子优化的人型支原体来源的adi基因片段,定向插入到pET-22b载体的NdeⅠ和XhoⅠ位点之间;重组的表达载体pET22b-adi转化大肠杆菌BL21(DE3),用IPTG诱导表达;表达产物通过透析复性后经DEAE离子交换层析纯化,SDS-PAGE检测表达及纯化结果 ;纯化的重组蛋白用二乙酰一肟-氨基硫脲比色法检测比活。结果成功诱导表达分子量为48 kDa的重组蛋白,目的产物以包涵体形式表达,表达量约为细胞总蛋白的15%。纯化后蛋白纯度达95%以上,比活为3 IU。结论本研究方法可制备得到较高纯度活性重组人型支原体ADI蛋白。Objective To investigate the expression and purification of arginine deiminase（ADI） from Mycoplasma hominis in E.coli.Methods An arginine deiminase gene from Mycoplasma hominis was condon-optimized and synthesized.After cloning into pET-22b vector,the pET-22b-adi expression vector was transformed into E.coli BL21 for expression by IPTG induction.Recombinant protein was refolded by dialysis and then purified with DEAE chromatography,followed by SDS-PAGE analysis.The activity of rADI was determined by diacetyl-mono-oxime method.Results The rADI with the molecular weight of 48 kDa was expressed in form of inclusion body and counted for about 15% of total protein.The purity of the purified rADI was more than 95%.Its activity was 3 IU.Conclusion Recombinant ADI with higher purity and activity was obtained in this method.

关 键 词：精氨酸脱亚胺酶 包涵体 复性 分离纯化 

分 类 号：R979.1[医药卫生—药品]