Expression and Purification of Human Tumor Necrosis FactorαFusing with Oligo- Histidine / 上海第二医科大学学报

ABSTRACT

ObjectiveExpression and purification of human tumor necrosis factorα(hTNFα)fusion protein with a stretch of six consecutive histidine residues(6×His) in E. coli.MethodshTNFα fusion proteins with 6×His at N and C terminus were expressed by using E. coli expression vectors pET- 28a(+) and pET-22b(+). The His6 tag allows the expression fusion protein purified in one step by immobilized metal Ni2+ chelation affinity chromatcgraphy 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 Ni2+ - IDA Sepharose 6B, 100ml induction culture had 0.4mg hTNFα-6×His fusion proteins. Its purifity reached 90 %. ConclusionThe purification expression product can possess TNF activity and reach 5.42×104U/mg.