Cloning, nucleotide sequence and expression of thioltransferase (glutaredoxin) cDNA from Schizosaccharomyces pombe.
Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previously purified a TTase from Schizosaccharomyces pombe and its molecular size was 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 was confirmed by Southern hybridization. The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein of 101 amino acids. The coding region of the original clone was transferred after the lac promoter of pUC13 vector for expression in E. coli, and simultaneously, a suitable Shine-Dalgarno (SD) sequence was added in front of the coding region by PCR. The two primers used for PCR also separately contained BamHI and HindIII restriction sites. The E. coli strain (A434) harboring the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.
Kim
H G
HG
Division of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon, Korea.
Cho
Y W
YW
Park
E H
EH
Park
S S
SS
Ahn
K S
KS
Lim
C J
CJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D054477:Glutaredoxins; D008969:Molecular Sequence Data; D010088:Oxidoreductases; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D012568:Schizosaccharomyces / Q000201:enzymology*; D016415:Sequence Alignment
D000596:Amino Acids; D005656:Fungal Proteins; D054477:Glutaredoxins; D011506:Proteins; D010088:Oxidoreductases