Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Dept. of Chemistry, Northwest University, No. 49 Chang'an North Road, Xi'an, China.
Biotechnol Prog. 2010 Jul-Aug;26(4):1073-9. doi: 10.1002/btpr.407.
Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC). This new method is applied to the refolding of a widely used model protein, urea-denatured/dithiothreitol-reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, beta-cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC-IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC-IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL(-1), the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC-IEC is a high efficient and low cost protein refolding method.
开发高效且低成本的蛋白质复性方法是生物技术领域的一个研究热点。人工分子伴侣（AMC）和蛋白质折叠液相色谱（PFLC）是近年来发展起来的两种有吸引力的复性方法。在本工作中，将 AMC 与 PFLC 的一个分支——离子交换色谱（IEC）集成，形成了一种新的复性方法，即人工分子伴侣-离子交换色谱（AMC-IEC）。该新方法应用于广泛使用的模型蛋白——尿素变性/二硫苏糖醇还原溶菌酶的复性。分别考察了影响溶菌酶复性的多种因素，如脲浓度、β-环糊精浓度、去污剂与蛋白质的摩尔比、流动相流速和去污剂类型，以优化通过 AMC-IEC 复性溶菌酶的条件。与正常的 IEC 复性方法相比，在所研究的初始蛋白质浓度范围内，通过 AMC-IEC 获得的溶菌酶的活性回收率要高得多。此外，对于高初始浓度的变性/还原溶菌酶，使用这种新开发的复性方法获得的活性回收率仍然相当高。当初始蛋白质浓度为 200mgmL(-1)时，活性回收率超过 60%。此外，在 AMC-IEC 过程中，色谱柱的寿命比在正常 IEC 复性过程中长得多。因此，AMC-IEC 是一种高效且低成本的蛋白质复性方法。
