Cloning, high level-expression and characterization of human lens thioltransferase.
Polymerase chain reaction (PCR) primers, directed against the nucleotide sequence of pig liver thioltransferase (PLTT) were used to amplify human lens thioltransferase (HLTT) from a pool of human lens cDNA. The 520 bp PCR fragment obtained was cloned unidirectionally into pCR 3.1-Uni vector and sequenced. The cDNA sequence of the lens thioltransferase had 98% and 87% homology to pig liver and human placental thioltransferases (TTase) respectively. Nhe1 and EcoR1 fragment of the recombinant PCR 3.1-Uni vector was subcloned in pET 23a Expression vector. High level expression of HLTT was accomplished in Escherichia coli and the expressed protein was characterized by immunoblot analysis with anti PLTT and N-terminal amino acid sequence analysis. The recombinant enzyme efficiently dethiolated protein thiol mixed disulfides conjugated to both cystine (PSSC) and glutathione (PSSG) and had a significant dehydroascorbate reductase activity. Human lens thioltransferase thus displayed structural and functional characteristics identical to pig liver and human placental thioltransferases.
Raghavachari
N
N
Center for Biotechnology, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, NE, 68583-0905, USA.
Qiao
F
F
Shinohara
T
T
Kikuchi
T
T
Lou
M F
MF
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D002999:Clone Cells; D003459:Crystallins / Q000378:metabolism; D054477:Glutaredoxins; D006801:Humans; D007908:Lens, Crystalline / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011490:Protein Disulfide Reductase (Glutathione)
D003459:Crystallins; D054477:Glutaredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); C020666:glutathione dehydrogenase (ascorbate)