CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

Thioredoxin (TRX)isanoxidoreductaseen zyme,withamolecularweightof 1 2kD .Itwasi dentifiedoriginallyinE .coliasanhydrogendonorforribonucleotidereductaseanddeoxyribonucle otidesynthesis1] .Thegeneofhumanthioredoxin(hTRX)islocatedonchromosome 3 p1 1 p1 2 ,withafulllengthof 1 3Kb .Theopenreadingframe ( 3 1 5nucleotideslong)codedforaproteinof 1 0 4aminoacids2 ,3] .ManyfunctionsofTRXhavebeenre ported,whichincludethefollowing :TheTRXsystem (whichincludesHADPHasaprotondonor,TRXreductase ,…

CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT-PCR cloning from 143 (TK-) human osteosarcoma cell. The amplified products were cloned into pGEM-T Easy vector and sequenced. Then the expressed vector pBV220-hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE-Sephadex A-50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.