体外重折叠核糖核酸酶A及其荧光结构表征  

Refolding of ribonuclease A in vitro and conformational characterization by spectrofluorimetry

作　　者：谭培生[1] 罗曼[1] 盛清[2] 关怡新[1] 姚善泾[1] 

机构地区：[1]浙江大学化学工程与生物工程学系,浙江杭州310027 [2]浙江理工大学生命科学学院,浙江杭州310018

出　　处：《浙江大学学报（农业与生命科学版）》2006年第5期483-488,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(20476093)

摘　　要：核糖核酸酶A(RNase A)作为蛋白质体外重折叠研究的模型蛋白,考察了稀释复性过程中脲浓度、稀释倍数、氧化还原环境(GSH浓度和GSH/GSSG比例)、pH值、温度以及聚集抑制剂的添加对该酶活性回收率的影响.在变性蛋白浓度为100μg?mL-1,复性液含有2 mol?L-1脲,GSH浓度2 mmol?L-1,GSH/GSSG比为4∶1,pH8.0,并添加终浓0.1%的PEG,转速为150 r?min-1,温度37℃的条件下复性,RNase A的活力回收率可达70%以上.利用荧光分析法对RNase A复性过程的结构变化进行了表征,在此基础上对RNase A体外重折叠过程蛋白质分子的构象变化以及活性中心的形成过程进行了阐述.Ribonuclease A （RNase A） was employed as a model protein to understand the formation of correct three-dimensional structure in the refolding of recombinant proteins in vitro. Important parameters such as the concentration of urea, dilution fold, redox environment （the concentration of GSH and the ratio of GSH to GSSG）, pH, temperature, and inhibitor of aggregation were investigated to demonstrate their effects on the activity recovery during refolding. The activity recovery was up to 70% under optimal condition as follows： 100 μg.mL^-1 denatured RNase A, refolding buffer containing of 2 mol. L^-1 urea, 2 mmol.L^-1 GSH and 0.1% PEG6000, the ratio GSH to GSSG 4 ： 1, pHS. 0, shaking speed at 150 r. min^-1 , 37℃ . Then, the mechanism of the refolding process of RNase A was characterized using spectrofluorimetry, which results give some supports to presume the conformational changes of RNase A along with the in vitro refolding proceeding.

关 键 词：核糖核酸酶A 稀释复性 荧光分析法 分子结构 

分 类 号：Q518.4[生物学—生物化学]