Optimized heterologous expression of glutathione reductase from cyanobacterium Anabaena PCC 7120 and characterization of the recombinant protein

Glutathione reductase (GR) from the cyanobacterium Anabaena PCC 7120 was he terologously expressed in Escherichia coli SG5. Silent random mutations wer e introduced in the 5' region of DNA encoding the enzyme in order to genera te a high-level expression clone. To maximize protein expression, the cultu re conditions were also optimized. In the high-level expression clones sele cted, E. coli-preferred codons were selectively used at certain, positions. Under the optimal expression conditions, a yield of 17 mg recombinant prot ein per liter was obtained, which is about 10-fold higher than that of the wild-type enzyme. A hexahistidine tag was added at the C-terminal of the pr otein in order to allow IMAC affinity purification. This strategy simplifie d the purification process and provided a homogeneous enzyme for functional characterization. Anabaena GR uses NADPH as a co-enzyme, like most of the GRs from other sources, but the K-M values for NADPH and GSSG are higher th an those of enzymes previously studied. The Anabaena enzyme also shows sign ificant activity when NADH is used as a reductant. (C) 1999 Academic Press.