高效弱阳离子交换色谱法对脲还原变性溶菌酶的折叠研究

Study on Refolding of Urea-Reduced/Denatured Lysozyme by High-Performance Weak-cation Exchange Chromatography

用高效弱阳离子交换色谱(HPWCX)对脲还原变性溶菌酶(Lys)进行了复性研究.在流动相中脲浓度固定为4.0mol?L-1和选用对天然态蛋白有稳定作用的硫酸铵为盐或置换剂时,在蛋白浓度为15.0～50.0mg?mL-1时,HPWCX法比稀释法活性回收率高.为了提高Lys的质量及活性回收率对所用色谱条件进行了优化研究,当蛋白起始浓度为20.0mg?mL-1时,Lys的质量回收率和活性收率分别为97.8%和95.4%.表明此种方法简便且有可能对其他还原变性蛋白的复性具有通用性.

The refolding of the urea-reduced/denatured lysozyme (Lys) by high-performance weak cation exchange chromatography was reported in this paper. With the presence of a fixed concentration of urea of 4.0 mol center dot L-1 and ammonium sulphate as salt or displacer having the stabilizing role to the structure of native protein molecules in the mobile phase employed, and when the Lys concentration was 15.0 similar to 50.0 mg center dot mL(-1), the bioactivity recovery by the presented method was higher than that by usual dilution method. The optimization of chromatographic conditions for obtaining the high recoveries of both mass and bioactivity of Lys was investigated in detail. The recoveries of both mass and bioactivity could be raised up to 97.8% and 95.4% respectively, as the Lys concentration in sample solution was 20.0 mg center dot mL(-1). The advantages of the presented method are simple operation, and high recoveries of both mass and bioactivity of Lys refolding, and thus it may possibly become a common method for many kinds of proteins.