Abstract
Recombinant rat squalene epoxidase (rSE) was expressed in E. coli and purified to an apparent homogeneity. This expression system was constructed using squalene epoxidase (SE) cDNA in which nucleotides coding 99 amino acids in the N-terminal were deleted and nucleotides coding hexa-histidine in the C-terminal were added. Purification was carried out using Ni-chelate affinity agarose and Cibacron Blue Sepharose column chromatography. Purification was achieved 100-fold over the crude E. coli extract with a yield of about 50%. The purified enzyme demonstrated a single band on SDS-polyacrylamide gel electrophoresis. The enzyme showed no distinct absorption spectrum in the visible regions. The properties of rSE were compared with those of rat liver microsomal SE. The requirement of the co-factors, the S105 fraction or Triton X-100, and NADPH-cytochrome c reductase, the pH dependency for enzyme activity, and the sensitivity to NB-598 seen with both enzymes suggest that rSE has properties very similar to rat microsomal SE. 2,3-Oxi-dosqualene (OSQ) and 2,3;22,23-dioxidosqualene (DOSQ) were formed by rSE in a completely reconstituted system. It is suggested that recombinant squalene epoxidase catalyzes the conversion of squalene to 2,3-oxidosqualene and of 2,3-oxidosqualene to 2,3;22,23-dioxidosqualene.
Highlights
Recombinant rat squalene epoxidasewas expressed in E. coli and purified to an apparent homogeneity
Triton X-100, and 8 p~ ["Hlsqualene (20000 dpm) disSimvastatin was obtained from Merck Sharp & Dohme persed in 0.075% Tween 80
An exceedingly low level of activity and the instability of squalene epoxidase (SE) make it difficult to purify the enzyme from rat liver microsomes
Summary
Recombinant rat squalene epoxidase (rSE)was expressed in E. coli and purified to an apparent homogeneity. Squalene epoxidase (SE) [EC 1.14.99.71,a membraneassociated enzyme in the middle stage of the sterol biosynthetic pathway, catalyzes the conversion of squalene to 2,3-oxidosqualene.SEwas purified to homogeneity by us [1, 2] Bai and Prestwich [3] reported the partial purification of pig liver SE and the effects of several substrate analog on enzymatic activity. Cholesterol feeding decreased hepatic SE activity, but the administration of an HMG-CoA reductase inhibitor and cholestyramine increased enzyme activity in rat. These results show that SE is regulated by the intracellular level of cholesterol. We compared the enzymatic properties of recombinant squalene epoxidase (rSE) with those of rat liver microsomal SE. We investigated whether rSE could form DOSQ in a completely reconstituted system
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