High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.
The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.
Sastre
J
J
U-99 INSERM, Hôpital Henri Mondor, Créteil, France.
Siegrist
S
S
Bulle
F
F
Asensi
M
M
Baik
J H
JH
Pawlak
A
A
Guellaën
G
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D004273:DNA, Neoplasm / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism* / Q000821:virology*; D013329:Structure-Activity Relationship; D005723:gamma-Glutamyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D004273:DNA, Neoplasm; D007527:Isoenzymes; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase