人类CYP8B1在大肠杆菌中的异源表达及全细胞活性检测

Heterologous expression and whole-cell activity assay of human CYP8B1 in Escherichia coli

摘要:

背景 据报道，抑制人类细胞色素P450 8B1 (cytochrome P450 8B1, CYP8B1)可以调节胆汁酸代谢，进而调节糖脂代谢，是2型糖尿病、肥胖症、非酒精性脂肪肝等代谢性疾病的一种潜在治疗策略，然而目前关于CYP8B1抑制剂的研究进展却非常缓慢，这可能是由于现有的靶向CYP8B1活性小分子测试体系难以实现中高通量筛选。目的 构建共表达人类CYP8B1与细胞色素P450还原酶(cytochrome P450 reductase, CPR)的大肠杆菌(Escherichia coli)系统，并筛选具有高效12α-羟化酶催化活性的菌株，旨在为建立CYP8B1的中高通量活性检测体系提供有效方法。方法 首先，基于双顺反子或双表达载体策略，以中高拷贝质粒pET-17b或高拷贝质粒pRSFDuet-1为载体，构建共表达CYP8B1与CPR的重组质粒；随后，将pGro7/pGro12 (可表达分子伴侣蛋白groES-groEL)和构建所得的共表达质粒先后转化不同的宿主菌株C41(DE3)、C43(DE3)、BL21-CodonPlus(DE3)-RIPL、Rosetta(DE3)和GSsetta(DE3)，构建不同的重组表达菌株。重组菌株经发酵后，以7α-羟基胆固醇(7α-hydroxy-4-cholesten-3-one, 7α-HCO)为CYP8B1底物，采用反相高效液相色谱(reverse phase-high performance liquid chromatography, RP-HPLC)检测法测定其全细胞催化活性，最终通过活性检测结果对比确定最优反应条件及表达系统。结果 我们成功构建了人类CYP8B1大肠杆菌的异源表达系统。分别以中高拷贝数质粒pET-17b和高拷贝数质粒pRSFDuet-1为载体，以分子伴侣蛋白groES-groEL表达质粒pGro7和pGro12，以C41(DE3)、C43(DE3)、BL21-CodonPlus(DE3)-RIPL、Rosetta(DE3)和GSsetta(DE3)作为宿主菌株，构建得到了CYP8B1的重组表达菌株12株。通过比较其全细胞活性，确定CYP8B1的最优表达系统为重组菌株C43-pRSF-tCYP8B1-tCPR，并且最优反应条件为：反应体系中不添加多黏菌素B，以二甲基亚砜(dimethyl sulfoxide, DMSO)作为底物7α-HCO的溶剂。结论 本研究构建的大肠杆菌表达系统可高效异源表达人类CYP8B1，并具备显著的全细胞催化活性。

Abstract:

Background Studies have shown that inhibiting human cytochrome P450 8B1 (CYP8B1) can regulate bile acid metabolism and further influence glucose and lipid homeostasis, representing a potential therapeutic strategy for metabolic diseases such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease. However, the development of CYP8B1 inhibitors remains slow, possibly due to the inability of existing small-molecule screening systems targeting CYP8B1 activity to achieve medium-to-high-throughput screening.Objective This study constructed an Escherichia coli system co-expressing human CYP8B1 and cytochrome P450 reductase (CPR) and identified the strains with high catalytic activity of 12α-hydroxylase, aiming to establish a medium-to-high-throughput screening system for CYP8B1 activity.Methods First, on the basis of bicistronic or dual-expression vector strategies, recombinant plasmids co-expressing CYP8B1 and CPR were constructed with the medium-to-high-copy plasmid pET-17b or the high-copy plasmid pRSFDuet-1 as vectors. Subsequently, pGro7/pGro12 (capable of expressing the molecular chaperone proteins groES-groEL) and the constructed co-expression plasmids were successively transformed into different host strains C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) to construct different recombinant expression strains. After fermentation of the recombinant strains, 7α-hydroxy-4-cholesten-3-one (7α-HCO) was used as the substrate for CYP8B1, and reverse phase-high performance liquid chromatography (RP-HPLC) was employed to measure the whole-cell catalytic activity. Finally, the optimal reaction conditions and expression system were determined by comparing the activity assay results.Results We successfully constructed a heterologous expression system of human CYP8B1 in E. coli. With the medium-to-high-copy plasmid pET-17b and the high-copy plasmid pRSFDuet-1 as vectors, along with plasmids pGro7 and pGro12 for expressing the molecular chaperone proteins groES-groEL, 12 recombinant expression strains of CYP8B1 were constructed in five host strains: C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3). By comparing their whole-cell activities, we identified the recombinant strain C43-pRSF-tCYP8B1-tCPR as the optimal expression system for CYP8B1, with the optimal reaction conditions being no polymyxin B added to the reaction system and dimethyl sulfoxide (DMSO) as the solvent for the substrate 7α-HCO.Conclusion The engineered E. coli system enables effective heterologous expression of human CYP8B1 with robust whole-cell catalytic activity.