枯草芽孢杆菌硫氧还蛋白还原酶的原核表达及活性分析

硫氧还蛋白还原酶(TrxR)是一种NADPH依赖的、含硒的黄素蛋白,在细胞增殖和分化、维持细胞内外氧化还原平衡中起重要作用。通过基因特异性引物从枯草芽孢杆菌BS04菌株中扩增TrxR编码基因,亚克隆至pET-28a(+)原核表达载体并转化大肠埃希菌BL21(DE3),经过IPTG诱导表达后采用Ni离子亲和层析进行重组蛋白纯化及活性分析。结果表明,枯草芽孢杆菌BS04菌株的TrxR基因全长1 011 bp,编码336个氨基酸,与已知枯草芽孢杆菌TrxR氨基酸同源性达99%。SDS-PAGE电泳和Western-blotting分析发现,重组蛋白分子量约37 ku,与预期相符。DTNB还原法测定TrxR活性及酶动力学常数结果表明,其对DTNB具有较高的还原活性,其K_m和K_cat值分别为3.06 mmol·L^-1和324.71 min^-1。该研究结果为进一步了解TrxR的功能提供了参考依据。

Prokaryotic expression and activity analysis of thioredoxin reductase from Bacillus subtilis

NADPH dependent thioredoxin reductase is a flavoprotein containing selenium, which plays an important role in cell proliferation and differentiation and maintaining cell oxidation-reduction equilibrium. The TrxR gene from Bacillus subtilis was amplified and subcloned into pET-28a(+) expression vector. Subsequently, the plasimid was transformed into Escherichia coli BL21(DE3). And then expression and purification of TrxR were performed. Results showed that the TrxR gene from B. subtilis strain BS04 was 1 011 bp in length, which encoding 336 amino acids; the amino acid identity between the gene we cloned and the sequence deposited in GenBank was about 99%. SDS-PAGE and Western-blotting analysis suggested that the molecular weight of the fusion protein was about 37 ku which was in accord with the estimated. Moreover, the kinetic parameters K_m and K_cat of the recombinant protein were determined to be 3.06 mmol·L^-1 and 324.71 min^-1 using DTNB assay. All above results provide good opportunity for our further study on the gene function.