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

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.