不同来源胆盐水解酶基因在大肠杆菌中的表达

Expression of Bile Salt Hydrolase Genes from Two Different Origins in Escherichia coli Rosetta

根据GenBank上报道的两歧双歧杆菌ATCC 29521的胆盐水解酶基因(BSH)序列和嗜酸乳杆菌NCFM的胆盐水解酶基因(BSHA和BSHB)序列,设计引物通过PCR扩增获得BSH基因,将其连接到表达载体pET28a(+),构建pET-BSH表达质粒,经IPTG诱导表达后,聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明在分子质量大小约40、43、45kD处有预期条带出现,初步表明蛋白表达成功。重组菌产生的胆盐水解酶水解甘氨胆酸钠产生的甘氨酸经茚三酮比色法分析,表明该重组的胆盐水解酶具有水解活性。

Bile salt hydrolase （BSH） gene of Bifidobacterium bifidum ATCC 29521 and Lactobacillus acidophilus NCFM was amplified by gradient PCR using genome DNA as template and cloned into pET28a （＋） expression vector. The expression vector was then transfected into E. coli Rosetta and expression of BSH was induced by IPTG. SDS-PAGE analysis showed the presence of three target proteins estimated to be 40, 43 kD and 45 kD, respectively. Ninhydrin analysis revealed that the recombinant BSH hydrolyzed sodium glycocholate to glycine, showing its hydrolysis activity on conjugated bile salts.