The thioredoxin system from Streptomyces coelicolor

Abstract

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.