核受体PPARγ-LBD在原核系统的最适表达及纯化  

Expression and purification of the nuclear regulator PPARγ-LBD in E.coli

作　　者：曹廷兵[1] 叶治家[1] 彭家和[1] 巩燕[1] 江渝[1] 

机构地区：[1]第三军医大学基础医学部生物化学与分子生物学教研室,重庆400038

出　　处：《第三军医大学学报》2003年第13期1146-1148,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目 ( 30 0 70 358) ;重庆市科技攻关项目( 2 0 0 12 6 )

摘　　要：目的　探索诱导PPARγ LBD的最适表达条件 ,提高可溶性PPARγ LBD蛋白在大肠杆菌中的表达量 ,然后用Ni2 + NTA离子交换树脂对表达蛋白进行分离纯化。方法　PPARγ LBD在大肠杆菌BL2 1(DE3 )中进行诱导表达时 ,以不同IPTG浓度、不同温度 ( 3 7℃ ,3 0℃ ,2 0℃ ) ,确定可溶性PPARγ LBD蛋白表达的最适条件 ;然后用Ni2 + NTA离子交换树脂进行分离纯化 ,其产物进行SDS PAGE纯度分析和Westernblotting鉴定。结果　建立了重组蛋白PPARγ LBD的最适诱导条件 ,即 0 .5mmol LIPTG浓度 ,2 0℃诱导 14h其可溶性表达蛋白的量最高 ;纯化产物经SDS PAGE纯度分析大于 90 %以上 ;Westernblotting检测 ,纯化产物为PPARγ LBD目的蛋白 ,其分子质量约 3 4× 10 3。结论　重组蛋白PPARγ LBD在E .coli中进行了成功表达和纯化 ,并且降低温度可提高可溶性蛋白PPARγ LBD表达量达 5 0Objective To find out the best expression condition to induce the increased output of the soluble PPARγ LBD protein in E.coli and to purify the expressed PPARγ LBD protein. Methods PPARγ LBD was induced to express in E.coli by means of different IPTG concentrations and different culture temperatures, then it was isolated and purified by Ni 2+ NTA ion exchange resin. The expressed PPARγ LBD was analyzed by SDS PAGE and identified by Western blotting. Results The best expression condition for recombinant protein PPARγ LBD was established: the maximum output of the soluble PPARγ LBD protein could be induced by 0.5 mmol/L IPTG at 20 ℃ after culture for 14 h. SDS PAGE revealed that the purity of the purified product was above 90%. Western blot analysis showed that the purified product was the objective protein of PPARγ LBD and its molecular weight was about 34×10 3 . Conclusion Recombinant protein PPARγ LBD is expressed successfully in E.coli and the output of soluble PPARγ LBD can be increased by 50% at temperature lower than that of normal induction.

关 键 词：PPARΓ PPARγ-LBD 表达 纯化 

分 类 号：R392.11[医药卫生—免疫学] R394-33[医药卫生—基础医学]