Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.
A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, MA.
D A
DA
Roth
A
A
Rehemtulla
R J
RJ
Kaufman
C T
CT
Walsh
B
B
Furie
B C
BC
Furie
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D019735:Carbon-Carbon Ligases; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors; D007700:Kinetics; D008025:Ligases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009036:Moths; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014812:Vitamin K / Q000494:pharmacology
D011994:Recombinant Proteins; D014812:Vitamin K; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase