Nagumo A, Kamei T, Sakakibara J, Ono T
Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd., Tsukuba, Japan.
J Lipid Res. 1995 Jul;36(7):1489-97.
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.
重组大鼠鲨烯环氧酶（rSE）在大肠杆菌中表达并纯化至表观均一性。该表达系统是利用鲨烯环氧酶（SE）cDNA构建的，其中N端编码99个氨基酸的核苷酸被删除，C端添加了编码六聚组氨酸的核苷酸。使用镍螯合亲和琼脂糖和Cibacron Blue琼脂糖柱色谱进行纯化。纯化后的产物比大肠杆菌粗提物提高了100倍，产率约为50%。纯化后的酶在SDS-聚丙烯酰胺凝胶电泳上显示出单一条带。该酶在可见光区域没有明显的吸收光谱。将rSE的性质与大鼠肝微粒体SE的性质进行了比较。两种酶对辅因子、S105组分或Triton X-100以及NADPH-细胞色素c还原酶的需求、酶活性的pH依赖性以及对NB-598的敏感性表明，rSE具有与大鼠微粒体SE非常相似的性质。在完全重构的系统中，rSE可生成2,3-氧化鲨烯（OSQ）和2,3；22,23-二氧化鲨烯（DOSQ）。提示重组鲨烯环氧酶催化鲨烯向2,3-氧化鲨烯的转化以及2,3-氧化鲨烯向2,3；22,23-二氧化鲨烯的转化。
