Expression, Purification And Site-directed Mutagenesis Of Thioredoxin System From Acidithiobacillus Ferrooxidans

Thioredoxin system constitutes of thioredoxin(Trx),thioredoxin reductase(TrxR),β-Nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt(NADPH).They all contain disulfide active site sequence of-Cys-Gly-Pro-Cys-.Thioredoxin system can stabilize the cells environment,regulate cell growth and signal transduction processes to protect cells from reactive oxygen species damage caused by external stimuli such as virus infection,ionizing radiation;It is also a reductant in many cellular reactions such as ribonucleotide reduction,and play an important role in the regulation of protein-protein expression,protein-nucleic acid expression.It is a widespread in nature system.Thioredoxin(Trx) is widespread presence in animals,plants,fungi and microbes,with a molecular mass of 12 kDa.It is an important protein for regulating enzymes activity,play a key role in redox carriers through the reversible thiol-disulfide redox change in a variety of reactions,and plays several key roles in maintaining the redox environment of the cell.The gene of thioredoxin from A.ferrooxidans ATCC23270 was cloned,overexpressed and purified by one-step affinity chromatography. We constructed the mutant expression plasmid(C33A,C33S, C36A,C36S) of the protein using site-directed mutagenesis.The UV-Vis scanning and activity test results confirmed that residues Cys33,Cys36 of thioredoxin were active-site residues.Thioredoxin reductase(TrxR) is a NADPH-dependent enzyme, contains FAD domain.It is a member of pyridine nucleotide-disulfide oxidoreductase family.The primary function of thioredoxin reductase is to catalyse the reduction of the small redox protern thioredoxin by NADPH,and maintain the reduced state of Trx.The gene of thioredoxin reductase from A.ferrooxidans ATCC23270 was cloned,expressed and purified by one-step affinity chromatography.The molecular mass of the recombinant protein was 35kDa,containing FAD.We constructed the mutant expression plasmid(C33A,C33S,C36A,C36S) of the protein using site-directed mutagenesis.The UV-Vis scanning and activity test results confirmed that residues Cys142,Cys145 of thioredoxin reductase were active-site residues.