胆固醇配基色谱在蛋白质复性中的应用

以三聚氯氰为连接臂,采用两步法将疏水配基胆固醇修饰到交联琼脂糖（Sepharose）介质上,构建了胆固醇配基色谱。以溶菌酶（Lys）为模型蛋白,考察了胆固醇配基色谱的复性效果,并研究了流速、上样质量浓度和上样量对色谱复性效果的影响,确定了溶菌酶复性的最佳方法。研究结果表明,当溶菌酶上样量为1.00 mL,介质含0.25 mg溶菌酶时,在0.02 mL/min操作流速下进行吸附-洗脱复性,其最终蛋白收率和活性收率分别为96.3%和91.4%,有效促进了溶菌酶的复性。

Application of Immobilized Cholesterol Column Chromatography in Protein Refolding

Cholesterol modified Sepharose matrix was prepared by a two-step method with cyanuric chloride as a linker. A refolding chromatography with immobilized cholesterol was studied for the reactivation of denaturedreduced lysozyme and the optimal method for protein refolding was established. Furthermore, the effects of flow rate, initial protein concentration and loading mass on refolding efficiency were investigated. The results showed that when the loading mass of Lysozyme was 0.25 mg/mL of the support, 96.3 % protein recovery and 91.4% activity yield were obtained by adsorption-elutlon way at flow rate of 0.02 mL/min. Denatured-reduced lysozyme was effectively refolded into the active form.