This paper describes the cloning, purification and characterization of thioredoxin (TrxA) and thioredoxin reductase (TrxR) from bacterial strain Streptomyces coelicolor . The genes of S. coelicolor encoding TrxA and TrxR were amplified by polymerase chain reaction, inserted into pET expression vector and used to overexpress these proteins in Escherichia coli . TrxA and TrxR were produced as the hexahistidine fusion proteins and were recovered from the cytoplasm as the soluble proteins. The activity of the purified recombinant proteins was demonstrated. The activity of TrxA was shown by efficient reduction of insulin and activity of NADPH-dependent TrxR was revealed by catalyses of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reduction reaction. The reduction reaction was fully dependent upon the presence of TrxA as intermediate electron carrier with the pH optimum 7.5 and the temperature optimum 29 degrees C. Km value of TrxR for TrxA was 0.217 +/- 0.02 microM.

PDB Reference: TrxA, 1t00, r1t00sf.Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disul®de reductases. In turn, they are reduced by electrons obtained from the NADPH-containing thioredoxin reductase. Thioredoxins have been isolated and characterized from a large number of organisms. The Grampositive bacterium Streptomyces coelicolor contains three thioredoxins that are involved in unknown biological processes. trxA from S. coelicolor was cloned and expressed in Escherichia coli and the protein puri®ed and crystallized using the hanging-drop method of vapour diffusion. The crystal structure of thioredoxin A has been determined at 1.5 A Ê resolution using a synchrotronradiation source. The protein reveals a thioredoxin-like fold with a typical CXXC active site. The crystal exhibits the symmetry of space group P2 1 2 1 2, with unit-cell parameters a = 43.6, b = 71.8, c = 33.2 A Ê .

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