Cloning, nucleotide sequence and expression of thioltransferase (glutaredoxin) cDNA from Schizosaccharomyces pombe

Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme th at catalyzes the reduction of a variety of disulfide compounds, including p rotein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previousl y purified a TTase from Schizosaccharomyces pombe and, its molecular size w as determined. In the present study, a cDNA coding TTase was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization, which- was constructed in a plasmid vector pGAD GH, and its corresponding insert w as confirmed by Southern hybridization, The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein o f 101 amino acids. The coding region of the original clone was transferred after the the promoter of pUC13 vector for expression in E. coli, and simul taneously, a suitable Shine-Dalgarno (SD) sequence was added in front of th e coding region by PCR, The two primers used for PCR also separately contai ned BamHI and HindIII restriction sites. The E. coli strain (A434) harborin g the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.