Investigation Of The Refolding Of Reduced/Denatured Ribonuclease A By Liquid Chromatography

The stoichiometric displacement parameters Z value of Lys in different molecular states were investigated firstly and RNase A refolding by ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) was studied.The thesis includes five parts as the follows:1. Review: Protein folding is a subject of fundamental and practical importance. A fundamental refolding strategy was introduced. All kinds of methods and development based on the strategy were reviewed.2. Based on the stoichiometric displacement theory for retention (SDT-R), the parameters Z and log/ were used to characterize the molecular conformational changes of lysozyme (Lys) for different molecular conformation states (pseudo-native, urea-unfolded, urea-reduced-unfolded) under various urea concentrations in weak cation exchange chromatography (WCX). The retention of the three molecular conformations of Lys totally follow the SDT-R .The Z values of the Lys in its pseudo-native state decreased with the increasing the concentration of urea in the mobile phase and was the biggest of the three molecular conformational states, while its corresponding log/ was the least one. Both Z and log/ of the Lys in their pseudo-native and urea-unfolded states were closed with each other. The changes of Z of Lys in the three molecular conformational states with urea concentration in mobile phase were found to bediscontinuously. The relationship between the Z value and bioactivity recovery of Lys under different urea concentrations was also investigated.3. Oxidative refolding of the denatured/reduced Ribonuclease A (RNase A) was investigated by using weak cation exchange chromatography (WCX) with the presence of reduced and oxidized glutathione in the mobile phase employed. Effects of urea concentration and the kind of salt on the renaturation ofreduced/denatured RNase A were investigated. It was found that the renaturationyield was significantly related to the urea concentration, urea concentration of 1.0-2.0mol/L in the mobile phase was found to increase the bioactivity recovery. The retention mechanism of the reduced RNase A on the WCX column was proved to be a mixed mode of ion-exchange and hydrophobic interaction. With the comparison of three kinds of stationary phase of WCX with the different matrixes, such as silica, sepharose and polymer, it was found every one had its own advantage. With experimental optimization for RNase A refolding, a considerably high bioactivity yield 84.7% was obtained even though the initial concentration of RNase A was raised up to 30.0 mg/ml by using of silica matrix column. The effects of the composition of mobile phase, urea concentration, pH, flow rate and the refolding time on the RNase A refolding were investigated.4. Reduced /denatured RNase A was refolded by using hydrophobic interaction chromatography (HIC). The highest bioactivity yield, 86.4% was obtained from the weekly hydrophobicity of HIC column, when the loading RNase A concentration was 3.0mg/mL. The bioactivity yield decreased with the increase of the loading protein concentration. The contribution of the composition of the mobile phase, urea concentration, pH, flow rate, and refolding time to RNase A refolding were also investigated .5. Refolding of reduced/denatured RNase A was compared by using size exclusion chromatography (SEC). In the presence of reduced and oxidized glutathione in mobile phase is necessary. With a linear decreased urea gradient urea gradient SEC also enhanced the yield of protein refolding, it provided a suitable environment to RNase A refolding .The flow rate of mobile phase was not found to have any important effects on the bioactivity yield of RNase A.