Yao, Ya-FengWeng, Yih-MingHu, Hui-YuLin, Long-Liu2006-12-092009-05-272006-12-092009-05-272006-12-09Yao YF, Weng YM, Hu HY, Lin LL. Overexpression of a recombinant gamma-glutamyltranspeptidase from Escherichia coli Novablue. Indian Journal of Biochemistry & Biophysics. 2006 Dec; 43(6): 345-50http://imsear.searo.who.int/handle/123456789/28783A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene, lacking the first 48-bp coding sequence for part of the signal sequence, was amplified by polymerase chain reaction (PCR) and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 21 kDa respectively by SDS-PAGE, indicating the precursor EcGGT still undergoes the post-translational processing even in the truncation of signal sequence. His6-tagged EcGGT migrated relative to the molecular mass of approximately 120 kDa and its heterodimeric structure was confirmed by a native-PAGE gel.engEscherichia coli --enzymologyRecombinant Proteins --biosynthesisgamma-Glutamyltransferase --biosynthesisJournal Article