利用荧光偏振方法的IDOL/LDLR蛋白相互作用评价体系的建立

Establishment of IDOL/LDLR protein-protein interaction system based on a fluorescence polarization assay

目的 利用荧光偏振方法建立体外低密度脂蛋白受体的诱导型降解因子(IDOL)/低密度脂蛋白受体(LDLR)的蛋白相互作用评价体系。方法 通过PCR扩增获得人IDOL蛋白全长cDNA,并与pET-30a(+)原核表达质粒进行重组,转化至大肠杆菌系统进行表达并纯化。将FITC荧光标记的LDLR多肽片段与His-IDOL共同孵育,利用荧光偏振方法,分别对多肽浓度、蛋白浓度以及反应时间进行优化,建立体外IDOL/LDLR的蛋白相互作用评价体系。结果 将pET-30a-IDOL重组质粒进行大肠杆菌原核表达,获得人全长IDOL蛋白。将His-IDOL重组蛋白进行His Trap柱亲和纯化,并与FITC-LDLR进行反应,最终确定500 nmol/L FITC-LDLR和2μmol/L His-IDOL于25℃、100 r/min振荡、避光孵育1 h为IDOL/LDLR相互作用最适反应体系。结论 利用荧光偏振方法成功构建了IDOL/LDLR蛋白相互作用评价体系,该体系为基于IDOL/LDLR相互作用的抗动脉粥样硬化的小分子抑制剂的开发和评价提供了实验基础。

Objective The identification system of IDOL interacting with LDLR in vitro was established using the fluorescence polarization assay.Methods The full-length cDNA of human IDOL was amplified by PCR and fused with pET-30a(+) prokaryotic expression plasmid.The recombinant plasmid was transformed into E.coli and the His-tagged IDOL protein was expressed and purified. The FITC fluorescent-labeled LDLR peptide was co-incubated with His-IDOL under different conditions, and the fluorescence polarization method was adopted in this assay. To establish the evaluation system of IDOL interacting with LDLR in vitro, the appropriate conditions were explored in this assay, including concentrations of polypeptide, protein concentrations and reaction time,respectively.Results The recombinant plasmid pET-30a-IDOL was induced with 0.5 mmol/L IPTG at 15 ℃ and 100 r/min overnight in order to obtain the full-length cDNA of human IDOL. The recombinant His-IDOL protein was purified with a HisTrap affinity column and co-incubated with FITC-LDLR. Finally, 500 nmol/L FITC-LDLR and 2 μmol/L His-IDOL incubating for 1 h without light at 25 ℃,shaking for 100 r/min, were considered to be the optimal reaction condition of the IDOL/LDLR interaction assay.Conclusion In this study, the IDOL/LDLR interaction system is successfully constructed using the fluorescence polarization method, which provides experimental basis for the development and evaluation of anti-atherosclerosis small molecule inhibitors based on IDOL/LDLR interaction.