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目的探讨通过金属螯合亲和层析的方法获得高纯度的人内皮细胞高表达脂多糖相关因子1(EOLA1)蛋白的可行性。方法采用蛋白抽提试剂破菌方法抽提包涵体蛋白,初步纯化后在变性条件下用组氨酸标签结合树脂进行亲和层析,以透析方法复性,对纯化样品进行聚丙烯酰胺凝胶电泳及免疫印迹法、肽质量指纹谱鉴定。结果EOLA1蛋白在大肠杆菌中的表达主要以包涵体形式存在,初步纯化的包涵体中蛋白纯度>75%,亲和层析纯化后获得的蛋白纯度高达90%以上,肽质量指纹谱分析目的蛋白与理论蛋白肽段覆盖率较高。结论所应用的EOLA1蛋白纯化和复性方法简便有效,能够获得足量的高纯度EOLA1蛋白,有助于下一步制备EOLA1单克隆抗体及对相关基因进行功能研究。
OBJECTIVE: To investigate the feasibility of high-level expression of lipopolysaccharide related factor 1 (EOLA1) protein in human umbilical vein endothelial cells by metal chelate affinity chromatography. Methods Inclusion body protein was extracted by the method of protein extraction reagent. The purified protein was purified by affinity chromatography using histidine tag and resin after denaturation, renaturation was performed by dialysis method. The purified sample was subjected to polyacrylamide gel Electrophoresis and immunoblotting, peptide mass fingerprinting identification. Results The expression of EOLA1 protein in E. coli mainly existed in the form of inclusion body. The protein purity of the purified inclusion body was> 75%, and the purity of the purified protein was over 90% by affinity chromatography. The peptide mass fingerprinting Peptides and theoretical peptide coverage higher. Conclusion The EOLA1 protein purification and renaturation method is simple and effective, and can obtain sufficient amount of high purity EOLA1 protein, which is helpful for further preparation of EOLA1 monoclonal antibody and functional study of related genes.