色谱

色谱 ›› 2010, Vol. 28 ›› Issue (08): 786-789.DOI: 10.3724/SP.J.1123.2010.00786

• 研究论文 • 上一篇    下一篇

还原变性核糖核酸酶在疏水性液-固界面上的复性

毕晶, 白泉*, 王军, 王骊丽   

  1. 西北大学合成与天然功能分子化学教育部重点实验室, 西北大学现代分离科学研究所, 现代分离科学陕西省重点实验室, 陕西 西安 710069
  • 收稿日期:2010-04-08 修回日期:2010-05-31 出版日期:2010-08-28 发布日期:2010-08-28
  • 通讯作者: 白泉,教授,博士生导师,主要研究方向为生物大分子的分离纯化.
  • 基金资助:

    国家“863”计划项目(No. 2006AA02Z227)和陕西省重点实验室重点科研项目(05JS62).

Refolding of reduced/denatured RNase A on the hydrophobic liquid-solid interface

BI Jing, BAI Quan*, WANG Jun, WANG Lili   

  1. Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Sciences, Key Laboratory of Separation Science in Shaanxi Province, Northwest University, Xi’an 710069, China
  • Received:2010-04-08 Revised:2010-05-31 Online:2010-08-28 Published:2010-08-28

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442

被引次数

2

摘要： 采用疏水相互作用色谱(HIC)对还原变性核糖核酸酶A (RNase A)在疏水性液-固界面上的复性进行了研究。详细讨论了流动相中脲的浓度、还原型谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)的比例、流动相pH和变性蛋白质浓度对还原变性RNase A复性效率和质量回收率的影响。结果表明,在最优化的复性条件(流动相中含有2.0 mol/L脲,GSH/GSSG的浓度比为8:1,流动相pH为8.0)下,还原变性RNase A能完全复性。当变性蛋白质质量浓度为5.0 mg/mL时,还原脲变性RNase A的活性回收率和质量回收率分别为98.0%和61.9%,还原胍变性RNase A分别为100.1%和66.8%。研究表明HIC是还原变性蛋白质复性的有力工具之一,可为蛋白质复性研究提供新方法和新思路。

关键词: 蛋白质复性, 核糖核酸酶A, 还原变性, 疏水作用色谱

Abstract: The renaturation of the reduced/denatured RNase A on the hydrophobic liquid-solid interface was investigated using hydrophobic interaction chromatography (HIC). The effects of urea concentrations, the ratios of reduced and oxidized glutathiones (GSH and GSSG), the pH of mobile phase and protein concentrations on the refolding efficiency and mass recovery of the reduced/denatured RNase A were investigated in detail. The results indicated that the reduced/denatured RNase A can be refolded completely under the optimized conditions of pH 8.0, 2.0 mol/L urea and the concentration ratio of GSH/GSSG of 8:1 in mobile phase. When the denatured protein was at the concentration of 5.0 mg/mL, the bioactivity efficiency and mass recoveries were 98.0% and 61.9% for 8.0 mol/L urea-denatured RNase A, respectively; and 100.1% and 66.8% for 7.0 mol/L guanidine hydrochloride (GuaHCl)-denatured RNase A, respectively. It proves that HIC is a powerful tool and new approach for protein refolding.

Key words: hydrophobic interaction chromatography (HIC), reduced/denatured, RNase A, protein renaturation