Cloning, expression and characterization of human thioltransferase (glutaredoxin) in E. coli.

PCR primers were designed from the known amino acid (aa) sequence for human red blood cell thioltransferase (hRBC TTase) and the known cDNA sequence for pig liver TTase (82% homologous) and used to amplify thioltransferase from a pool of human brain cDNAs. The PCR product was inserted into the pKK233-2 expression vector. The DNA sequence of the insert agreed with the aa sequence. High level expression of the enzyme was accomplished in E. coli, and Western blot analysis confirmed its identity. Recombinant TTase displayed catalytic properties indistinguishable from natural hRBC TTase.