透析法体外复性抗凝溶栓双功能HV12p-rPA融合蛋白  

The Renaturation of A Fusion Protein HV12p-rPA by Dialysis in Vitro

作　　者：梁兰[1] 余蓉[1] 邓璇[1] 李磊[1] 吴梧桐[2] 

机构地区：[1]四川大学华西药学院,四川成都610041 [2]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2009年第2期140-143,共4页Pharmaceutical Biotechnology

基　　金：生化工程国家重点实验室开放基金资助项目(2005KT2-23);教育部博士点基金资助项目(20040316005)

摘　　要：探索表达于大肠杆菌中的抗凝溶栓双功能水蛭素12肽和瑞替普酶融合蛋白(The fusion protein of 12 peptide of hirudin and reteplase,HV12p-rPA)的体外复性方法。采用直接透析复性和氧化复性结合透析复性两种方式,并分析复性时间、温度、适宜的氧化-还原体系比例对复性率的影响。分别测定其体外抗凝活性和纤溶活性,确定复性效果。结果显示:将变性溶解的HV12p-rPA在含8mol/L脲,0.05mmol/LGSSG,0.7mol/LL-Arg的50mmol/LGly-NaOH(pH9.20)缓冲液中于25℃氧化复性6h后,再在含0.5mol/LL-Arg,1mmol/L胱氨酸,2mmol/L半胱氨酸的20mmol/LGly-NaOH(pH9.20)缓冲液中于4℃进行梯度脲浓度透析,每隔8h换液一次,透析48h,可获得具有抗凝活性为730ATU/mg,纤溶活性为19768IU/mg的可溶性蛋白质。The purpose of the study is to explore the refolding protocal for the fusion protein of 12 peptide of hirudin and reteplase（HV12p-rPA）expressed in E. coli in vitro. The inclusion body was renatured by dialysis or a combination of oxidation and dialysis. Several factors affecting refolding yield were analyzed,including refolding time, temperature and redox environment. The refolding effect was evaluated by fibrinolytie activity and anticoagulant activity respectively. The result showed that denatured HV12p-rPA should be firstly oxidized in 50 mmol/L Gly-NaOH（pHg. 20）buffer containing 8 mol/L urea,0.05 mol/L GSSG,0. 7 mol/L L-Arg for 6h, then dialyzed at 4℃ under the condition of 0. 5mol/L L- Arg, 1 mmol/L Cystine, 2 mmol/L Cysteine, 20 mmol/L GIy-NaOH（pH9. 20）buffer containing a concentration gradient of urea. The solution outside the dialysis tubing is replaced every 8h. The refolding time is 48 h. Then the soluble protein with anticoagulant activity of 730 ATU/mg and fibrinolytic activity of 19768 IU/mg could be acquired.

关 键 词：HV12p-rPA 包涵体 透析 氧化 复性 

分 类 号：Q503[生物学—生物化学]