摘要
应用pET28(含T_7lac启动子和His6-tag)质粒,将人γTNFβ融合基因与之组建成pETγLT表达质粒,并转化于E.coliBL21(DE3).本文对pETγLT/ BL21(DE3)工程菌的His6-γTNFβ重组产物进行了诱导表达,分离纯化的研究.结果表明,该工程菌用LB培养基在37℃扩增至OD_(590)为0.5左右,加IPTG(终浓度为1mmol/L)诱导5小时,此时His6-γTNFβ的表达量可占菌体总蛋白的45%.重组产物绝大多数以IBs形式存在于菌体中.工程菌经反复超声破碎、高速离心,得IBs粗制品后,再经含2mol/L脲的缓冲液洗涤可得到相对纯净的IBs.然后用7mol/L脲变性溶解IBs,离心取上清,进行Ni^(2+)-Sepharose 6B柱一步法亲和层析,可得到电泳纯度为95%的His6-γTNFβ,经稀释复性和用凝血酶切去His6-tag的产物,其细胞毒活性为(1.2～2.0)×10~7U/mgp,抗病毒活性为(6.0～6.6)×10~6U/map.
The E. coli BL21(DE3) strain bearing the plasmid(T_7lac/His6-tag) with the hγTNFβrecombinant gene was grown in LB medium containing SOμg/ml kanamycin, at 37 ℃ , up to the late logarithmic phase(OD590 ~ 0.5)then induced with IPTG (final concentration lmmol/L)for 5 hours. SDS-PAGE analysis revealed that expression level of the product(His6-γTNFβwas up to 45% of the total bacterial proteins and was mainly as insoluble inclusion bodies ( IBs) . After cell lysis, the IBs was separated by centrifugation, dissolved in 7mol/L urea, then purified by Ni column ( Ni^(2+) -Sepharose 6B) . And the purity of more than 95% and the recovery of about 90% were obtained. The purified product was refolded with the renaturation buffer under low temperafure( < 10 ℃) ,and allowed for removed of the His6-tag at N terminus using the appropriate protease (thrombin). The specific cytotoxic activity and the specific antiviral activity of the renaturation product were (l .2 ~ 2.0) x 10~7U/mgp and (6.0 -6.6) ×10~6U/mgp respectively.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
1998年第4期256-260,共5页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金(39670815)资助项目。
关键词
人IFNγ/TNFβ
融合蛋白
分离纯化
hγTNFβ(Human IFNγ/TNFβ)
fusion protein
purification and renaturation
