蛋白质二硫键异构酶a3的表达纯化、活性测定与小分子抑制剂的筛选

Expression,purification,activity determination of protein disulfide bond isomerase a3 and small molecule inhibitors screening

目的 通过大肠杆菌表达并纯化蛋白质二硫键异构酶a3(PDIA3)为筛选其小分子抑制剂奠定基础。方法 通过RT-PCR获得PDIA3基因,构建重组质粒pET28a-PDIA3,转化大肠杆菌BL21(DE3),IPTG诱导表达,Ni-NTA树脂亲和层析获得PDIA3,SDS-PAGE鉴定纯度,酶标仪测定活性,Specs商业化合物库虚拟筛选PDIA3的抑制物,胰岛素比浊法对抑制物进行抑酶活性测定。结果 克隆序列与基因库序列一致。获得的纯化PDIA3比活力为4.17 U/mg,PDIA3具有催化二硫键断裂的能力,筛选到的化合物去甲氧基姜黄素和41974954对PDIA3有抑制作用。结论 成功表达并纯化出PDIA3,筛选到的去甲氧基姜黄素和化合物41974954对PDIA3有抑制作用,为PDI蛋白抑制剂和抗血栓药物的研发奠定了基础。

Objective To develop the small molecular inhibitors screening method targeted protein disulfide isomerase a3(PDIA3) by recombinant expression and purification of PDIA3 in E.coli.Methods The PDIA3 gene was amplified by RT-PCR,and transferred to pET28 a vector by enzyme digestion and ligation method.The recombinant plasmid pET28-PDIA3 was transformed into E.coli BL21(DE3),induced to express by Isopropyl β-D-1-thiogalactopyranoside(IPTG),recombinant PDIA3 was obtained by Ni-NTA resin affinity chromatography.The protein purity was identified by SDS-PAGE,and the bioactivity was determined by Insulin turbidimetry assay.The virtual screening of the compound library targeted PDIA3 was conducted by the MOE software,and the screened compounds were tested for enzyme inhibitory activity by insulin turbidimetry.Results The Sanger sequencing result showed that the cloned sequence is consistent with the PDIA3 sequence in GenBank.The recombinant PDIA3 had a high purity and a specific activity of 4.17 U/mg.The recombinant PDIA3 showed good bioactivity to catalyze the cleavage of disulfide bonds.The demethoxycurcumin and compound 41974954 screened by MOE reveals inhibitory effects on PDIA3 cleavage activity.Conclusion PDIA3 was expressed and purified successfully in E.coli,and inhibitory effect of the demethoxycurcumin and compound 41974954 by virtual screening was confirmed using recombinant PDIA3.In summary,the data obtained in this study will aid the development of inhibitors targeted PDI protein and antithrombotic drugs.