乳杆菌bsh基因的高效表达及高活性酶蛋白获得

High expression of bsh gene from Lactobacillus and purification of enzyme protein

目的探讨乳杆菌bsh基因的高效表达及胆盐水解酶(BSH)的高活性酶蛋白获得。方法从本实验室保存的12株乳杆菌菌株中,通过Ca2+沉淀法初筛获得具BSH活性的菌株,根据GenBank上公布的16S rRNA同源性最高的乳杆菌的BSH全基因序列,设计特异性引物,PCR扩增获得bsh基因并克隆至表达载体pET-28α,构建BL21-pET-28α-bsh原核表达体系。经IPTG诱导后,对表达产物进行SDS-PAGE分析及Western blot验证。结果研究表明在分子质量大小约35、37 kD处有预期条带出现,初步表明BSH蛋白表达成功。进而对表达各条件进行优化,最佳酶活条件为IPTG浓度0.1 mmol/L、诱导pH 6.5、诱导温度37℃、诱导时间1 h。经镍柱亲和层析纯化制备BSH制剂,邻苯三酚红钼法蛋白浓度测定为1446 mg/dL,茚三酮法测酶活性最高可达21248.27 U/(g·prot),为纯化前的635.4倍。结论乳杆菌bsh基因可溶性表达成功且高酶活BSH纯化获取为进一步的研究和药物转化奠定基础。

Objective To explore the high expression of Lactobacillus bsh gene and purification of enzyme protein of bile salt hydrolase(BSH).Methods Ca2+precipitation method was used on 12 Lactobacillus strains preserved in our laboratory to preliminarily screen the trains with BSH activity.Enzyme coupling colorimetric method was used to calculate the absorption of cholesterol.Based on the BSH gene sequence of Lactobacillus which was published on GenBank and had the highest homology with the experimental 16 S rRNA,specific primer was designed.PCR amplification was performed to obtain the bsh gene which was then cloned onto its expressing carrier pET-28αto construct the BL21-pET-28α-bsh prokaryotic expression system.After IPTG induction,SDS-PAGE analysis and Western blot verification were performed on the expressing product.Results The expected bands appeared at 35 kD and 37 kD,indicating successful expression of BSH protein.Then the expression conditions were optimized,resulting in an optimal IPTG concentration of 0.1 mmol/L,induction temperature of 37℃,inducing pH of 6.5,and induction time of 1 hour.The BSH protein was purified using nickel column affinity chromatography.The protein concentration was 1446 mg/dL by catechol red molybdenum method;the highest enzyme activity of BSH was 21248.27 U/(g·prot)by ninhydrin method,which was 635.4 times the value before purification.Conclusion The successful of soluble expression of bsh gene and acquisition of high active BSH will help build a foundation for further mechanism research and drug transformation.