不同复性条件对重组人组织纤溶酶原激活剂复性的影响

不同复性条件对重组人组织纤溶酶原激活剂复性的影响 Effects of Different Conditions on Recombinant Human Tissue-Type Plasminogen Activator Renaturation 吴正辉 WU Zhenghui1 1 first-author 郜金荣 GAO Jinrong1 1 佘应龙 SHE Yinglong1 1 叶力 YE Li1 1 叶林柏 YE Linbai1 1 武汉大学生命科学学院/病毒学国家重点实验室 武汉大学生命科学学院/病毒学国家重点实验室 College of Life Sciences,Wuhan University/State key Laboratory of Virology College of Life Sciences,Wuhan University/State key Laboratory of Virology 大肠杆菌生产菌株诱导表达产生的无活性重组人组织纤溶酶原激活剂（tissue-type plasminogen acti-vator,tPA）包涵体蛋白,溶解于尿素中变性后进行复性.通过单因子试验和正交试验考察了不同复性条件下透析复性对tPA包涵体蛋白复性效率的影响.结果表明蛋白质浓度、复性温度、pH值、复性介质、还原剂浓度对tPA的复性效果都有不同程度的影响,当0.5 g/L tPA包涵体蛋白溶解于pH 9.5的7 mol/L的尿素,加入5 mmol/Lβ-巯基乙醇,在20℃、pH 8.0的双蒸水条件下复性时,可以取得最高的复性效率（2.0×107IU/g）.通过对稀释复性、透析复性和凝胶过滤层析复性3种方法比较,建立了适宜工业化生产tPA的复性方法. The tPA（tissue-type plasminogen activator） crystal was expressed from an E.coli engineering strain and dissolved in urea.Then effects on the renaturation of tPA crystal under different conditions were analyzed with single factor test and orthogonal test.Our results indicated that tPA concentration,temperature,pH,renaturation buffers as well as the concentration of reducing agent β-mercaptoethanol（β-ME） all had significant effects on tPA renaturation.The optimal procedure for tPA renaturation is 0.5 mg/ml crystal being dissolved and denatured in 7 mol/L urea at pH9.5 with 5 mmol/L β-ME,then renatured by dialysis with double distilled water（pH 8.0） at 20 ℃.By this way the highest tPA activity（2.0×107 IU/g） can be obtained.The comparison of efficiency in tPA renaturation by dilution,dialysis and gel column chromatography suggests the proper tPA renaturation procedures for large-scale production. tPA蛋白 包涵体 复性 正交试验 recombinant tPA inclusion bodies renaturation orthogonal test Q78 国家十五攻关项目（2001BA749C）;湖北省重大项目（2001AA304B01） 2008-04-01 2021-04-01 4