苜蓿中华根瘤菌与耐盐有关基因rstA在大肠杆菌中的高效表达及其产物的纯化  

The expression of gene related to salt tolerance fromSinorhizobium meliloti 042BM inEscherichia coli and purification of its fusion protein

作　　者：葛世超[1] 王磊[1] 李小红[1] 亓苏伟[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院微生物学系农业部微生物资源及其应用重点实验室,北京100094

出　　处：《微生物学报》2005年第3期455-458,共4页Acta Microbiologica Sinica

基　　金：国家"973项目"( 0 0 1CB10 890 5 );国家"863计划"( 2 0 0 3AA2 4115 0 );欧盟科技合作项目 (ICA4 CT 2 0 0 1 10 0 5 6)~~

摘　　要：苜蓿中华根瘤菌(Sinorhizobiummeliloti) 0 4 2BM与耐盐有关的1 9kbDNA片段含有两个开放阅读框,采用PCR方法分别将它们扩增,连接到穿梭质粒上,并进行了耐盐功能检测,证明其中的ORF2具有耐盐性,定名为rstA基因。将它分别克隆到表达载体pThio HisA、B和C上,构建成重组质粒pGSA、pGSB和pGSC ,转化大肠杆菌(Escherichiacoli)Top10后,经IPTG诱导,pGSA获得高效表达。表达蛋白占菌体总蛋白的36 % ,但大多数以包涵体形式存在。对表达产物依次进行ProBondTM树脂亲和纯化、饱和硫酸铵盐析,最后得到纯度为95 %的融合蛋白。SDS PAGE显示纯化的蛋白质为分子量4 3kD的单一蛋白带。A 1.9kb DNA fragment related to salt tolerance ofS.meliloti strain 042BM containing two open reading frames were obtained by PCR amplication and ligated into shuttle vector pBBR1-MCS2. The complementation experiment showed that ORF2 is related to salt tolerance and named asrstA gene. Then the gene was cloned into the expression vector pThio-HisA, B and C, respectively, and recombinant expression vectors pGSA, pGB and pGC were constructed,and transformed intoE.coli Top10. Inducing by IPTG and analyzing with SDS-PAGE, the fusion protein encoded by pGSA was obtained,and it is 36% content of whole cell protein. It was isolated and purified by affinity chromography on ProBond TM , and the inclusion body precipitated by saturated sulfate ammonium, and 95% purity of fusion protein was obtained. The final product displayed a single band with a corresponding molecular weight 43kD in SDS-PAGE, and was verified by the Western blot.

关 键 词：苜蓿中华根瘤菌 耐盐 表达 纯化 

分 类 号：Q943.2[生物学—植物学]