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 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.
Nagumo
A
A
Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd., Tsukuba, Japan.
Kamei
T
T
Sakakibara
J
J
Ono
T
T
D000818:Animals; D001483:Base Sequence; D001596:Benzylamines / Q000494:pharmacology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010105:Oxygenases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013185:Squalene / Q000031:analogs & derivatives / Q000378:metabolism; D050603:Squalene Monooxygenase; D013876:Thiophenes / Q000494:pharmacology
D001596:Benzylamines; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013876:Thiophenes; C066059:NB 598; C015171:2,3,22,23-dioxidosqualene; D013185:Squalene; D017830:Octoxynol; D010105:Oxygenases; D050603:Squalene Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase