Expression, purification, and characterization of the human squalene synthase: use of yeast and baculoviral systems.
We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral expression systems. Expression of human squalene synthase in yeast resulted in production of active enzyme in cellular lysates. The presence of the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting that structural differences in the yeast and human enzymes may affect localization or folding of the protein. Expression of the human enzyme in Sf-9 insect cells after infection with recombinant baculovirus encoding the human squalene synthase gene resulted in detection of substantial enzymatic activity in cell lysate preparations. Following extraction from the Sf-9 cells, the human enzyme was purified to near homogeneity utilizing a series of ion-exchange chromatography steps with an overall yield of purified protein of approximately 5 mg per liter of Sf-9 cell culture. The purified enzyme was characterized through steady-state kinetic and physical measurements and the kinetic constants are consistent with values observed for other squalene synthases. Zaragozic acid C was found to be a competitive inhibitor with respect to farnesyl pyrophosphate and has a Kis value of 250 pM (@ [NADPH] = 5 mM). Inhibition experiments with zaragozic acid C at low (approximately 0.5 x Km) and high (approximately 10 x Km) concentrations of NADPH indicated that the inhibitor does not bind in the enzyme's NADPH binding domain. These studies demonstrate that the human enzyme can be prepared from baculovirus-infected Sf-9 cells in a catalytically active configuration and in sufficient quantities to allow for further biochemical, kinetic, and structural characterization.
Soltis
D A
DA
Department of Atherosclerosis and Vascular Biology, Sandoz Pharmaceuticals Corporation, East Hanover, New Jersey 07936.
McMahon
G
G
Caplan
S L
SL
Dudas
D A
DA
Chamberlin
H A
HA
Vattay
A
A
Dottavio
D
D
Rucker
M L
ML
Engstrom
R G
RG
Cornell-Kennon
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002478:Cells, Cultured; D003001:Cloning, Molecular; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012717:Sesquiterpenes; D013045:Species Specificity; D018411:Spodoptera / Q000166:cytology
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C079090:zaragozic acid C; D009249:NADP; C004808:farnesyl pyrophosphate; D013186:Farnesyl-Diphosphate Farnesyltransferase