人γ干扰素/β肿瘤坏死因子融合蛋白His6融合表达及一步纯化

Over-expression of the His6-γ/TNF-β protein and single-step purification

将人γ干扰素 /β肿瘤坏死因子融合蛋白 ( hγTNF-β)重组基因克隆于表达载体 p ET2 8,构建成T7lac启动子控制下的 His6融合表达质粒 ,转化溶源菌 E.coli BL 2 1 ( DE3 ) .经 IPTG( 1 m M)诱导表达 ,阳性菌在 SDS-PAGE泳图上出现一条粗表达带 ,其分子量 ( 3 2 k Da)与目的蛋白 His6-γ TNF-β)理论分子量相符 .薄层扫描与溶解性分析显示 ,表达产物占菌体总蛋白的 4 5%以上 ,主要为不溶性的包涵体( IBs) .离心分离 IBs产物 ,溶于 7M尿素中 ,然后通过 Ni柱进行亲和层析 ,即可获得一步纯化的表达产物(纯度为 96%、回收率为 91 % ) .纯化产物再经复性缓冲液稀释复性 ,其细胞毒比活性与抗病毒比活性分别达到 1 .2× 1 0 7～ 2 .0× 1 0 7u/m gp和 6.6× 1 0 5～ 7.2× 1 0

The h IFN -γ/TNF-β fusion protein( hγTNF-β) recombinantgene was cloned to the expression vector p ET2 8,and a T7lac promoter-based fusion expression plasmid was constructed that directed the synthesis of hγTNF-β in E.coli as fusion with a stretch of six consecutive histidine residues( His6-tag) at N terminus.SDS-PAGE analysis revealed that with IPTG( 1 m M) induction the strain bearing the plasm id highly expresses the target protein ( 3 2 k D) which m olecular weight is sam e as the theory m olecular weight of His6-γ TNF-β,and expression level of the product( as insoluble inclusion bodies, IBs) is above 4 5% of the total bacterial proteins.After cell lysis,the IBs is pelleted by centrifugation, dissolves in 7M urea,then purifies in one step by Ni colum n( Ni2 + -sepharose 6B) .And the purity of m ore than 96% and the recovery of 91 % were obtained.The purified product was refolded under low temperature( <1 0℃ ) .The specific cytotoxic activity and the specific antiviral activity of the renaturation product are 1 .2× 1 0 7～ 2 .0× 1 0 7u/mgp and 6.6× 1 0 5～ 7.2× 1 0 5u/mgp respectively.