title: Overexpression of a recombinant gamma-glutamyltranspeptidase from Escherichia coli Novablue.
abstract: A 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.
authors.affiliation: Graduate Institute of Food Science, National Chiayi University, 300 University Road, 60083 Chiayi, Taiwan.
authors.forename: Ya-Feng
authors.initials: YF
authors.lastname: Yao
authors.forename: Yih-Ming
authors.initials: YM
authors.lastname: Weng
authors.forename: Hui-Yu
authors.initials: HY
authors.lastname: Hu
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase