重组人胰岛淀粉样多肽的制备及评价

Preparation and Evaluation of Recombinant Human Islet AmyloidPolypeptide(hIAPP)

人胰岛淀粉样多肽(hIAPP)的淀粉样变与2型糖尿病(T2D)的发生发展密切相关。为解决体外评价化合物抗hIAPP淀粉样变活性所需hIAPP存在化学合成困难、成本高、溶解性低和批次稳定性差等问题,本研究以蛛丝蛋白N端结构域突变体NT作为融合标签,构建大肠杆菌高效表达hIAPP体系。通过优化表达条件和分离纯化工艺,获得重组hIAPP(rhIAPP),并对rhIAPP二硫键形成、淀粉样变能力和淀粉样变产物的毒性进行检测。融合蛋白质以包涵体形式存在,经过尿素变性和亲和层析纯化得到融合蛋白质产量为70 mg/L发酵液,融合蛋白质经过复性、凝血酶切除融合标签和进一步纯化,最终得到rhIAPP产量为6 mg/L发酵液。检测显示,rhIAPP的巯基未形成二硫键。rhIAPP具有“S”型淀粉样变曲线,其形成的β-折叠结构远多于化学合成的hIAPP(chIAPP),化合物姜黄素能显著抑制2种来源hIAPP的淀粉样变。细胞毒检测表明,rhIAPP在与红细胞孵育过程中促进红细胞溶血。

The amyloidosis of human islet amyloid polypeptide(hIAPP)is closely related to the development and pathogenesis of type 2 diabetes(T2D).In this study,we constructed a highly efficient expression system of hIAPP in E.coli using a spider silk protein N-terminal domain mutant NT as a fusion tag to solve the problems of synthesis difficulty,high cost,low solubility,and poor stability of chemically synthesized hIAPP for in vitro evaluation of compounds anti-amyloid formation ability.The recombinant hIAPP was obtained by optimizing the expression conditions and purification processes.With the recombinant hIAPP,we tested the recombinant hIAPP(rhIAPP)for the disulfide bond formation,amyloidosis ability,and amyloidosis product cytotoxicity.The fusion protein was isolated from inclusion bodies and was purified using urea denaturation and affinity chromatography,yielding a 70 mg/L fermentation broth.After renaturing,thrombin cleaving the fusion label,and further purification,the rhIAPP gave a final yield of a 6 mg/L fermentation broth.It showed that the two sulfydryl groups did not form disulfide bonds.The rhIAPP amyloidosis displayed an“S”-shaped curve,and when compared to chemically synthesized hIAPP(chIAPP),the fibrils formed moreβ-folded structures,and both sources of hIAPP amyloid formation can be greatly suppressed by the substance curcumin.Meanwhile,cytotoxicity analysis revealed that rhIAPP promoted red blood cell hemolysis during incubation.