PC12细胞硫氧还蛋白cDNA的克隆及在大肠杆菌中的表达

Cloning of TRX cDNA from PC12 cells and expression in E.coli

硫氧还蛋白 (Thioredoxin ,TRX)是广泛存在于原核和真核细胞中的低分子量蛋白质 ,它含有保守的Cys Gly Pro Cys活性位点 ,作为多效性细胞因子而具有重要的生物学功能。从PC1 2细胞中提取总RNA ,经逆转录PCR(RT PCR)扩增出硫氧还蛋白cDNA并克隆到PUC1 8质粒上。序列分析表明 ,克隆所得序列与GenBank中大鼠硫氧还蛋白cDNA序列完全一致。将该基因亚克隆到表达质粒pQE30上 ,质粒pQE30 TRX在大肠杆菌M1 5中获得高效表达。带 6His的融合蛋白约占总菌体蛋白的 30 %。分析鉴定表明 ,纯化的融合蛋白质分子量是 1 4kD ,且具有二硫键还原酶活性。

Thioredoxin(TRX) is a low molecular weight protein found in both prokaryotic and eukaryotic cells and has a conserved Cys Gly Pro Cys active site..It plays an important biological role as a multifunctional protein. Total RNA was extracted from PC12 cells. The TRX cDNA obtained by RT PCR was cloned into PUC18vector. Sequence analysis verified that the sequence of the cloned cDNA is identical to that of rat TRX cDNA in Genbank.Then the TRX cDNA was subcloned into expression vector PQE30 and was highly expressed in Ecoli M15.The amount of 6His tag fusion protein took about 30% of total bacterial protein. Analysis demonstrated that the fusion protein has a thiodepedent reducing activity and its molecular weight is 14kD.