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Abstract 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 display the dual function of thioredoxin and thioredoxin reductase.
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Received: 17 June 2010
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