PURIFICATION AND CHARACTERIZATION OF RECOMBINANT SQUALENE EPOXIDASE

Recombinant rat squalene epoxidase (rSE) was expressed in E. coli and purified to an apparent homogeneity. This expression system was constr ucted using squalene epoxidase (SE) cDNA in which nucleotides coding 9 9 amino acids in the N-terminal were deleted and nucleotides coding he xahistidine 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. c oli extract with a yield of about 50%. The purified enzyme demonstrate d a single band on SDS-polyacrylamide gel electrophoresis. The enzyme showed no distinct absorption spectrum in the visible regions. The pro perties of rSE were compared with those of rat liver microsomal SE. Th e requirement of the co-factors, the S-105 fraction or Triton X-100, a nd NADPH-cytochrome c reductase, the pH dependency for enzyme activity , and the sensitivity to NB-598 seen with both enzymes suggest that rS E has properties very similar to rat microsomal SE. 2,3-Oxidosqualene (OSQ) and 2,3;23,23-dioxidosqualene (DOSQ) were formed by rSE in a com pletely reconstituted system. It is suggested that recombinant squalen e epoxidase catalyzes the conversion of squalene to 2,3-oxidosqualene and of 2,3-oxidosqualene to 2,3;22,23-dioxidosqualene.