小麦TaNADP-ME1基因在大肠杆菌中的融合表达及可溶蛋白纯化

The Fusion-expression of Wheat TaNADP-ME1 Gene in E. coli and Purification of Fusion Protein

NADP依赖的苹果酸酶(NADP-ME)是C4光合途径关键酶。为了确定TaNADP-ME1基因的功能,利用重组技术将前期克隆到的TaNADP-ME1基因构建到原核表达载体pET32a,双酶切和PCR鉴定阳性克隆,CaCl2法转化大肠杆菌BL21(DE3)pLysS,IPTG诱导融合蛋白表达,Ni2+-NTA琼脂糖亲和层析柱纯化融合蛋白。成功获得了重组原核表达载体pETE1,TaNADP-ME1基因在BL21(DE3)pLysS中得到了融合表达,SDS-PAGE表明,融合蛋白分子量为80 kDa,并成功纯化到融合蛋白。

NADP-dependent malic enzyme（NADP-ME）is a key enzyme in C4 photosynthesis,the objective is to construct the TaNADP-ME1 gene into prokaryotic expression vector,express fusion protein in E.coli and purify the fusion protein.The TaNADP-ME1 gene was constructed into expression vector pET32a by recombination technology,recombination plasmid was identified by digestion with restriction enzymes and PCR amplification,and transformed into BL21（DE3）pLysS by CaCl2 method,fusion protein was induced by IPTG and purified by Ni2＋-NTA agarose column.This research successfully acquired the recombination vector pETE1,and TaNADP-ME1 gene was accurately expressed in BL21（DE3）pLysS,SDS-PAGE revealed that the molecular weight of purified fusion protein was about 80 kDa and successfully acquired the fusion protein.This study laid a good foundation for identification the gene function of TaNADP-ME1 gene.