Establishment of a V79 transfected cell line highly producing recombinant human gamma-glutamyltransferase.
gamma-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 cell line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 cells. A cell line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this cell line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered cell line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
Oster
T
T
Centre du Médicament, URA CNRS 597, Nancy, France.
Visvikis
A
A
Thioudellet
C
C
Fournel-Gigleux
S
S
Wellman
M
M
Siest
G
G
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase