Prokaryotic expression and activity analysis of chloroplast NADPH dependent thioredoxin reductase from maize | 玉米叶绿体依赖NADPH硫氧还蛋白还原酶的原核表达及活性分析

Chino. 叶绿体依赖烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)的硫氧还蛋白还原酶含有还原酶结构域和硫氧还蛋白结构域，每个结构域含有两个催化的Cys残基。利用RT-PCR克隆了编码成熟的依赖NADPH硫氧还蛋白还原酶基因，分别将还原酶结构域以及硫氧还蛋白结构域活性中心两个催化的Cys残基定点突变成Ser残基，将野生型和突变型基因分别插入大肠杆菌(Escherichia coli)表达载体pET-28b，转化至大肠杆菌BL21(DE3)，表达的重组蛋白N端含有组氨酸标签。电泳检测显示野生型硫氧还蛋白还原酶的可溶性表达水平受诱导温度和共表达分子伴侣GroEL、GroES 和 GrpE影响，而蛋白突变体主要表达为包涵体。纯化的重组野生型蛋白SDS-PAGE上显示亚基分子量为52 kD，分别以DNTB(6,6'-Dinitro-3,3'-dithiodibenzoic acid)和胰岛素为底物，确定了硫氧还蛋白还原酶两个结构域的催化活性。本研究结果表明, 玉米NTRC在大肠杆菌可溶性表达,纯化的重组蛋白具有硫氧还蛋白和硫氧还蛋白还原酶活性。

Inglés. Nicotinamide adenine dinucleotide phosphate (NADPH) thioredoxin reductase from chloroplast contains reductase domain and thioredoxin domain. Each domain has the two catalytic cystein residues. Using RT-PCR method, the gene encoding the mature NADPH dependent thioredoxin reductase from chloroplast (NTRC) was cloned from maize(Zea mays) young leaves. The catalytic cysteine residues in reductase domain, and thioredoxin domain were mutated into serine residues, respectively. The wild and mutated genes were inserted individually into the expression vector pET-28b and the constructed vectors were transformed into Escherichia coli strain BL21 (DE3). The recombinant proteins were fused with histidine-tag at N terminus. SDS-PAGE analysis showed that the soluble expression level of the recombinant NTRC wild type was affected by the inducing temperature, and co-expression of molecular chaperones including GroEL, GroES and GrpE. The mutated proteins were expressed mainly as inclusion bodies. The recombinant NTRC wild type was purified by Ni-NTA affinity chromatography. The molecular weight of the NTRC subunit was estimated about 52 kD, as shown by SDS-PAGE. The activities for both domains of NTRC using DNTB and insulin as the substrates were determined respectively. The results indicate that maize NTRC is overexpressed in the soluble form in E. coli. The purified recombinant protein displays the dual function of thioredoxin and thioredoxin reductase.