Cloning and expression of the glutaredoxin ( grx) gene of Escherichia coli

Abstract

Two DNA segments, together comprising 1147 bp and containing the glutaredoxin (GRX) gene, grx, from Escherichia coli K-12 were cloned and characterized in M13mp9. The gene was identified by hybridization with synthetic oligodeoxyribonucleotide probes corresponding to parts of the amino acid (aa) sequence of GRX. The sequence of 255 bp comprising the GRX structural gene gave a deduced aa sequence identical to the directly determined one. The coding region is preceded by two possible ribosome-binding sites and three possible promoters with − 10 and − 35 regions as judged by homology to consensus sequences. The presence of a stable stem-loop structure, ΔG = −17.0 kcal, followed by six thymine bases indicates that the transcription of the grx gene is Rho-independently terminated. An over-representation of rare codons in the grx gene, as compared to the genes for thioredoxin (TRX) and highly expressed proteins, is suggested as one possible explanation for the large difference in the synthesis between TRX and GRX in wild-type E. coli cells. GRX production was amplified at least 100 fold in strain JM103[pEMBL9ECG] over that in wild-type E. coli cells. The protein purified from the overproducing strain was identical in aa composition and N-terminal aa sequence with the previously analyzed GRX protein.