Contribution Of Stationary Phase Of Weak-Cation Exchange Chromatography To Refolding Of Denatured/reducced Lysozyme

The selecting of eight kinds of column of weak-cation exchange chromatography (WCX) was firstly investigated for the reduced/denatured lysozyme (Lys) refolding. The column bonding 3-nitrophthalic anhydride was found to be the best. The parameters effecting on the reduced/denatured Lys were also studied.The thesis includes four parts as the follows:1. Review: Protein folding is a subject of fundamental and practical importance and liquid chromatography (LC) is one of the new methods. Contribution of stationary phase to protein refolding and disulfide bond formation was reviewed, and 76 references were cited.2. Using silica gel as matrix, many modified reactions to increase hydrophilicity were carried out by using polyethylene (PEG) with different molecular weights, such as PEG200, PEG400, PEG600, PEG 1000. The increases in the hydrophilicity of the stationary phase can reduce and avoid non-reversible adsorption of biopolymers. By evaluating the refolding of the reduced/denatured Lys with WCX column, PEG400 column was selected for the refolding of denatured/reduced Lys.3. In WCX, a little changes of structure and constitute of ligands will cause a great effect on the Lys refolding. The four kinds of ligands, 3-nitrophthalic anhydride, succinic anhydride, phalic anhydride, and phenylsuccinic anhydride as the end groups were bond to the employed WCX column. Their contributions to the refolding of the denatured/reduced Lys were also evaluated, and the first one of them was found to be the best. A new definition called ratio R(b/m) of mass recovery (Rm) and bioactivity recovery (Rb) was firstly presented. With the R(b/m) the effect from experimental conditions of either adsorption or Lys refolding by LC can be judged4. The refolding of the denatured/reduced Lys was investigated by using the WCX in the presence of the reduced and oxidized glutathione in the mobile phase employed. Effects of urea concentration and the kinds of salt on the renaturation of the reduced/denatured Lys were carefully studied. It was found that the Lys renaturation yield was significantly related to the urea concentration. When urea concentration was of 3.0molL-1 and (NH4)SO4 was as salt in the mobile phase, the bioactivity recovery of the renatured Lys was found to increase. The retention mechanism of the reduced Lys on the WCX column was proved to be a mixed mode of ion-exchange and hydrophobic interaction. With experimental optimization for Lys refolding, a considerably high bioactivity yield 95.38% was obtained, even though the initial concentration of Lys was raised up to 20.0 mg mL-1.