High-level expression of enzymatically active mature human gamma-glutamyltransferase in transgenic V79 Chinese hamster cells.
gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 cell line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 cells. We selected a cell line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this cell line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered cell line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
Visvikis
A
A
Centre du Médicament, Centre National de la Recherche Scientifique Unité Associée 597, Nancy, France.
Thioudellet
C
C
Oster
T
T
Fournel-Gigleux
S
S
Wellman
M
M
Siest
G
G
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase