还原变性核糖核酸酶的高效液相色谱复性  

Refolding of reduced/denatured ribonuclease by high performance liquid chromatography

作　　者：黄亚渝[1] 童卫[2] 郭立安[3] 耿信笃[4] 

机构地区：[1]第四军医大学西京医院血液科 [2]第四军医大学医教部,陕西西安710033 [3]第四军医大学药学系化学教研室,陕西西安710033 [4]西北大学化学系,陕西西安710069

出　　处：《第四军医大学学报》2005年第15期1423-1426,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30080007)

摘　　要：目的:研究还原变性的核糖核酸酶(RNase)在高效疏水作用色谱(HPHIC)及高效离子交换色谱(HPIEC)上的复性及影响因素.方法:在二硫苏糖醇(DTT)存在的条件下用盐酸胍(GuHCl)和尿素(Urea)将RNase变性还原,先在溶液中加入氧化型谷胱苷肽(GSSG)进行氧化,然后在流动相中有低浓度变性剂存在的条件下,用HPHIC,HPIEC及稀释法对其进行复性并测定其生物活性及回收率.结果:HPHIC和HPIEC对RNase的复性效率均高于稀释法,两者的色谱固定相对RNase的复性均有所贡献.在色谱流动相中加入低浓度的变性剂可以提高复性效率,改变GSSG的浓度对复性效率也有影响.结论:用HPHIC,HPIEC对RNase进行复性,不仅复性效率高,而且快速、简便,具有很好的应用前景.AIM： To investigate the refolding of the reduced/denatured ribonuclease （RNase） by high performance hydrophobic interaction chromatography （HPHIC）and high performance ion exchange chromatography（HPIEC） and to study the factors influencing its refolding.METHODS： The reduced/denatured RNase by guanidine hydrochloride or urea with high concentration in the presence of dithiotheitol （DTT） was first oxidized by oxidizedgluthatione （GSSG） in solution and then refolded by liquid chromatography. RESULTS： In the presence of low concentration of denaturing agent and a suitable concentration of GSSG in the mobile phrase, the bioactivity and mass recoveries of the refolded RNase increased. CONCLUSION：Compared with other elution methods, both HPHIC and HPIEC, with the advantages of high renaturation efficiency,fast process and simple operation, have good potentials in application.

关 键 词：核糖核酸酶 蛋白折叠 疏水作用色谱 离子交换色谱 

分 类 号：Q511[生物学—生物化学]