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 e...

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Bibliographic Details
Published in:	Archives of biochemistry and biophysics Vol. 316; no. 2; pp. 713 - 723
Main Authors:	Soltis, D.A., Mcmahon, G., Caplan, S.L., Dudas, D.A., Chamberlin, H.A., Vattay, A., Dottavio, D., Rucker, M.L., Engstrom, R.G., Cornellkennon, S.A., Boettcher, B.R.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-02-1995
Subjects:	Amino Acid Sequence Animals Baculoviridae - genetics Base Sequence Bridged Bicyclo Compounds - pharmacology Bridged Bicyclo Compounds, Heterocyclic Cells, Cultured Cloning, Molecular Farnesyl-Diphosphate Farnesyltransferase - antagonists & inhibitors Farnesyl-Diphosphate Farnesyltransferase - biosynthesis Farnesyl-Diphosphate Farnesyltransferase - genetics Farnesyl-Diphosphate Farnesyltransferase - metabolism Genetic Complementation Test Genetic Vectors - genetics Humans Molecular Sequence Data NADP - metabolism Polyisoprenyl Phosphates - metabolism Recombinant Proteins - biosynthesis Recombinant Proteins - metabolism Saccharomyces cerevisiae - genetics Sesquiterpenes Species Specificity Spodoptera - cytology
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Summary:	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 (∼0.5 × Km) and high (∼10 × 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-9861 1096-0384
DOI:	10.1006/abbi.1995.1095