Title of article :
Yeast Squalene Synthase: Expression, Purification, and Characterization of Soluble Recombinant Enzyme
Author/Authors :
Zhang، نويسنده , , D.L and Jennings، نويسنده , , S.M and Robinson، نويسنده , , G.W and Poulter، نويسنده , , C.D، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 1993
Pages :
11
From page :
133
To page :
143
Abstract :
Squalene synthase is the first pathway-specific enzyme in the cholesterol biosynthetic pathway. The gene (ERG9) encoding squalene synthase in Saccharomyces cerevisiae has been isolated and characterized (S. M. Jennings, Y. H. Tsay, T. M. Fisch, and G. W. Robinson, 1991, Proc. Natl. Acad. Sci. USA 88, 6038-6042: M. Fegueur, L. Richard, A. D. Charles, F. Karst, 1991, Curr. Genet. 20, 365-372). The structural gene for the enzyme was modified by the polymerase chain reaction to remove a hydrophobic C-terminal domain, and the open reading frame for the truncated protein was cloned into yeast and Escherichia coli expression vectors. In E. coli, over-expressed truncated squalene synthase was soluble and constituted 2-5% of total cellular protein. The recombinant enzyme was purified to >95% homogeneity in two steps by chromatography on hydroxyapatite and phenyl-Superose. Soluble truncated squalene synthase is monomeric and catalyzes the two-step conversion of farnesyl diphosphate (FPP) to squalene via presqualene diphosphate in the presence of Mg2+ and NADPH. The concentration of FPP needed for half-maximal activity was 40 μM. At higher concentrations, FPP was an inhibitor. The activity of squalene synthase was stimulated by detergents and reached a maximal value of kcat = 3.3 s−1 at 100 μM FPP in the presence of 1% (v/v) Tween 80.
Journal title :
Archives of Biochemistry and Biophysics
Serial Year :
1993
Journal title :
Archives of Biochemistry and Biophysics
Record number :
1450534
Link To Document :
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