摘要
重组羧肽酶原B在大肠杆菌中过量表达时形成包涵体,需要经过体 外变复性后才能获得生物活性．为了提高羧肽酶原B的复性效率,首先对包涵 体的溶解条件进行了优化．对比了极端pH条件、各种变性剂和一些表面活性剂 对包涵体的溶解效果;并且在较弱的碱性条件下得到了很好的包涵体溶解效果． 接着,通过对缓冲液中蛋白浓度、pH值、温度、氧化还原对(GSH:GSSG)比值 的定量分析,确定了复性液的基本成分;比较了不同浓度的尿素对防止聚集、提 高复性效率的影响;另外还对比了加样方式的影响,最终确定了重组羧肽酶原B 体外复性的最佳条件,即20 mmol／L Tris-HCl,pH=9．5,150μg／mL Pro- CPB concentration,1 mmol／L GSH,0．5 mol／L urea．复性效率比最初提高了3 倍左右．
Recomcombinant pro-Carboxypeptidase B formed insoluble inclusion bodies when overexpressed in Escherichia coli ,and it must be denatured and renatured in vitro before it acquired activity. Various buffers including denaturants, extreme pH, detergents and Tris buffer at high temperature were used to solubilize inclusion bodies and pH = 11 was proved to have good solubility. To increase the renaturation yield of denatured pro-Carboxypeptidase B,the basic renaturation conditions of pro-Carboxypeptidase B were decided through qualitative and quantitative analysis of denaturant concentration,pH,temperature, the ratio of reduced and oxidized reagent and dilution ways. The optimized renaturation buffer was as follows： 20 mmol/L Tris-HCl, pH = 9. 5, 150μg/mL Pro-CPB concentration, 1 mmol/L GSH,0.5 mol/L urea. The renaturation effect was three times than before.
出处
《分子科学学报》
CAS
CSCD
2005年第4期51-57,共7页
Journal of Molecular Science
基金
上海市重点学科资助项目
关键词
重组羧肽酶原B
变性
复性
pro-Carboxypeptidase B
denaturation
renaturation
