分枝杆菌Mycobacterium sp. NwIB-01 3-甾酮-9α-羟基化酶基因的克隆、异源表达及分离纯化

Cloning, heterologous expression and purification of a 3-ketosteroid-9α-hydroxylase (KSH) from Mycobacterium sp. NwIB-01

摘要:

3-甾酮-9α-羟基化酶(KSH)是微生物甾体降解途径中的关键酶, 红平红球菌在甾体药物制备中有重要价值。以本实验室从土壤中自行筛选的分枝杆菌Mycobacterium sp. NwIB-01为出发菌株, 利用Rhodococcus erythropolis SQ1已报道的ksh序列与已全基因组测序的分枝杆菌序列数据库进行比对分析, 根据同源基因设计简并引物获得部分ksh序列, 通过染色体步移扩增出全长ksh(命名为M.S.-ksh), 该基因与耻垢分枝杆菌M. smegmatis mc2155的ksh同源性为85%。构建pET32-ksh表达载体, 转化大肠杆菌BL21(DE3), 获得高表达重组转化子菌株, 经IPTG低温诱导, SDS- PAGE电泳分析, 目的蛋白主要为可溶性表达, 表达量占菌体总蛋白的30%以上, 用Ni2+亲和层析柱纯化, 纯度达90%以上。本研究为利用基因工程菌进行工业化生产甾体药物奠定了基础。

Abstract:

3-ketosteroid-9α-hydroxylase (KSH), a key enzyme in the microbial steroid degradation, is highly valuable for the production of some stroid drugs. Degenerate primers were designed by comparing the ksh from Rhodococcus erythropolis SQ1 and its homologous sequences in the reported genome of Mycobacteria. Subsequently, a gene fragment of KSH was cloned from Mycobacterium sp. NwIB-01, a sterol-transforming bacterium isolated from soil in our lab. According to the conservative sequence, the full-length 1188 bp gene encoding ksh (designated as M.S.-ksh) was obtained by chromosome walking, which showed 85% identity with the ksh of M. smegmatis mc2155. The heterologous expression of KSH was achieved in Escherichia coli BL21(DE3) using the pET-32a-c(+) vector system. The expressed KSH protein was mostly in soluble form after IPTG induction at 30oC and accounted for more than 30% of total bacterial proteins according to SDS-PAGE electrophoresis. The molecular mass of KSH was about 45 kD, which was exactly the size predicted. After Ni2+ affinity chromatography, the purity of the target protein was more than 90%. Our work will definitely contribute to the industrial production?of some steroid drugs by developing KSH genetically engineered bacteria.