Artificial Molecular Chaperon-Ion Exchange Chromatography Assisted Refolding Of Denatured/Reduced Proteins

High efficiency and low cost protein refolding methods are a highlighted research focus in downstream process of biotechnology. In this thesis, artificial molecular chaperon (AMC) and ion exchange chromatography (IEC) were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC) was developed. The refolding process of a model protein, urea-denatured/dithiothreitol (DTT)-reduced lysozyme, was investigated by this new method, and recombinant human granulocyte colony-stimulating factor (rhG-CSF) was renatured using this method. There were four parts in this thesis:1. Review: The recent developments in several aspects, such as significance of protein refolding, protein refolding method, protein folding liquid chromatography, separation and purification of recombinant human granulocyte colony-stimulating factor, were briefly reviewed. 97 references were cited in this chapter.2. Refolding of denatured/reduced lysozyme by artificial molecular chaperone-ion exchange chromatography. A novel protein refolding method, AMC-IEC, was developed by integrating AMC and IEC, and this new method was applied to the refolding of urea-denatured/DTT-reduced lysozyme. Many factors affecting the refolding of lysozyme, such as urea concentration,β-cyclodextrin (β-CD) concentration, pH, flow rate of mobile phase, molar ratios of detergent/lysozyme, types of detergents, were investigated respectively to optimize the refolding conditions of lysozyme by AMC-IEC. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations in this work. Moreover, the activity recovery was still very high for denatured/reduced lysozyme at high initial concentration when using this newly developed refolding method, when the initial protein concentration was 200 mg/mL, the activity efficiency was 63%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than during normal IEC refolding methods. Therefore, AMC-IEC is a high efficiency and low cost protein refolding method.3. Refolding of denatured/reduced lysozyme by one step-artificial molecular chaperone-ion exchange chromatography. Sodium lauroyl sarcosine (Sarkosyl, SLS) was used as a denaturant, a novel protein refolding method, which was proposed on the basis of one step-AMC and IEC, one step-AMC-IEC was used on the refolding of denatured/reduced lysozyme. Many factors affecting the refolding of lysozyme, such as concentrations of urea,β-CD, pH of mobile phase and mobile phase flow rate, were optimized respectively. When they were 3.0 mol/L, 8 mmol/L, 7.5, and 1.0 mL/min, respectively, the activity recovery were highest.4. Refolding of recombinant human granulocyte colony-stimulating factor by AMC-IEC. rhG-CSF inclusion bodies were solubilized by different methods, the result showed that 2% Sarkosyl solution could solubized the inclusion bodies efficiently.β-CD was used as a stripping agent, the rhG-CSF solubilized by Sarksoyl was refolded by using one step-AMC-IEC and AMC-IEC, respectively. The results indicated that mass recoveries of total proteins obtained by these two methods were comparable, and they both were higher than that obtained by IEC.