Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone-ion exchange chromatography.
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.
Wang
Chaozhan
C
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. czwang@nwu.edu.cn
Zhang
Qinming
Q
Cheng
Yan
Y
Wang
Lili
L
D002852:Chromatography, Ion Exchange / Q000379:methods*; D018832:Molecular Chaperones / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding
D018832:Molecular Chaperones; D009113:Muramidase