hTNFα寡聚组氨酸融合蛋白的表达  

Expression and Purification of Human Tumor Necrosis Factor α Fusing with Oligo-Histidine

作　　者：李彪[1] 朱承谟[1] 吴祥甫[2] 

机构地区：[1]上海第二医科大学瑞金医院核医学科,上海200025 [2]中科院上海生物化学研究所

出　　处：《上海第二医科大学学报》2000年第4期348-351,共4页Acta Universitatis Medicinalis Secondae Shanghai

摘　　要：目的利用大肠杆菌表达系统对人肿瘤坏死因子 (hTNFα)的寡聚组氨酸 (6×His)融合蛋白进行表达和纯化研究。　方法采用大肠杆菌表达系统的表达载体 pET - 2 8a(+)和 pET - 2 2b(+)表达了hTNFα 的 6×His融合蛋白 ,其 6×His分别位于融合蛋白的N和C端 ,并利用寡聚组氨酸与过渡态金属离子的高亲和力性质 ,经Ni2 +-IDASepharose 6B亲和柱对表达产物进行了纯化。　结果其表达量分别为菌体总蛋白的 45 %和 8%。前者在胞内以不溶性包涵体存在 ,未对表达产物作进一步纯化 ;后者在胞质空间以可溶性存在 ,经亲和纯化的hTNFα- 6×His纯度可达 90 %以上 ,得率为 0 .4mg/ 10 0ml,并对L92 9细胞具有杀伤的功能 ,其活力为 5 .42× 10 4 U/mg。　 结论表达产物经纯化后具有细胞毒的活性。Objective Expression and purification of human tumor necrosis factor α (hTNFα)fusion protein with a stretch of six consecutive histidine residues(6×His) in E.coli. Methods hTNFα fusion proteins with 6×His at N and C terminus were expressed by using E.coli expression vectors pET-28a(+) and pET-22b(+). The His 6 tag allows the expression fusion protein purified in one step by immobilized metal Ni 2+ chelation affinity chromatography in native state. Results The two construct expression vectors were expressed in E.coli respectively, the former with high level as insoluble protein, account for 45% of the total bacteria proteins and not purified; the later 8% as soluble protein, and characterized by SDS-PAGE, Westren-blot. By using affinity chromatography through Ni 2+ -IDA Sepharose 6B, 100ml induction culture had 0.4mg hTNFα-6×His fusion proteins. Its purifity reached 90%. Conclusion The purification expression product can possess TNF activity and reach 5.42×10 4U/mg.

关 键 词：肿瘤坏死因子 寡聚组氨酸 纯化 融合蛋白 表达 

分 类 号：R730.2[医药卫生—肿瘤]