| High performance liquid chromatography (HPLC) has been successfully applied to the separation of protein, refolding with purification denatured proteins, in which the surface properties of stationary phase play a key role. In this system, the protein separating and refolding at the liquid chromatography are accomplished by hydrophobic interactions, electrostatic interaction and specific interaction functional ligand. This study shows that the presence of plurality interactions between stationary phase and protein could promote separation of proteins while inhibiting protein aggregation as well as precipitation of denatured proteins to improve the efficiency of protein refolding. For example, a zwitterionic ion chromatography (ZIC) stationary phase with ligand of phosphorylcholine (PC) significantly improves its selectivity and separability so that three natural proteins can be separated from egg in a single step at pH 5.5. And the stationary phase with ligand of tryptophan can refold with simultaneous purification of recombinant human Delta-like1 in both hydrophobic interaction chromatography (HIC) and weak cation exchange chromatography (WCX). Therefore, this study selected 2-methacryloyloxyethyl phosphoryl choline (MPC) which could forms cell membrane structure as the ligand to synthesize a stationary phase of polymer brushes structure based on silica. As containing various functional groups (including positive charge, negative charge, hydrophobic alkyl chain), this ligand could provide electrostatic adsorption, electrostatic repulsion and hydrophobic interaction. Hence, this work will characterize the stationary phase with phosphorylcholine ligand (abbreviation:MPC stationary phase) including its retention features, separation properties and possible application.The dissertation consists of four sections:1. Literature reviewThe development of HPLC depends on the research of stationary phase. In addition to single chromatographic stationary phase, mixed mode chromatography has been widely used in the separation and purification of naturally active ingredients, proteomics and the refolding with simultaneous purification of recombinant protein. Due to two interactions provided by stationary phase, the mixed system has higher selectivity and protein loading. The HPLC stationary phase types, preparation methods and applications will be reviewed in this section.2. Synthesis and characterization of phosphorylcholine ligand stationary phaseResearch shows that, materials containing MPC functional groups have hydrophilic anion and cationic, can be highly hydrated and have negative surface charge in a wide pH range. Therefore, the stationary phase based on silica gel functionalized with MPC as ligand prepared by "thiol-ene" click reaction, has a structure of polymer brushes structure on the surface of stationary phase. With elemental analysis and X ray photoelectron spectroscopy, the results indicate that, MPC could successfully bonded to the surface of the stationary phase, with a ligand density of 1.26?mol/m2 and a dynamic adsorption of 51.5mg/g.3. Retention characterization and separation performance of phosphorylcholine ligand stationary phaseConfirmed by evaluating with different standard proteins and polar compounds, the MPC stationary phase has two liquid chromatography modes including WCX and HILIC. In detail, six standard proteins were completely separated in WCX mode, and five nucleosides were separated in HILIC mode with this stationary phase. The effects of pH and salt concentration on the separation of standard protein in WCX mode, and the effects of water volume fraction, pH, ionic strength on the separation of nucleosides in HILIC mode were further investigation. The results showed that the retention features on this stationary phase fully compliant with the laws of both chromatographic modes. The MPC stationary phase was employed in the separation with purification of egg white protein and the separation with determination of puerarin from Pueraria thomsonii. The results showed that the mass recovery and purity of Lysozyme can reached to 98.2% and 95% respectively. And HILIC efficiency was evaluated by determination of Puerarin in Pueraria thomsonii with the MPC stationary phase.4. Refolding with simultaneous purification of denatured protein by the phosphoryl- choline ligand stationary phaseFirstly, reduced/denatured lysozyme was used as the model protein to study the refolding with this MPC stationary phase in WCX mode. Effects of urea concentration, pH and GSH/GSSG on refolding of lysozyme were investigated. As a result, when the mobile phases contained 3.0mol/L urea,6:1 GSH/GSSG and pH 6.5, the mass recovery and the activity recovery of lysozyme were 91.9% and 94.1%, respectively. Then, the MPC stationary phase has been applied to refolding with simultaneous purification of recombinant human Notch ligand and Delta-like1 (rhDlll) expressed in Escherichia coli (E. coli). The results showed that when the mobile phases contained 3.0mol/L urea, the mass recovery and purity of rhDlll could reach to 63.4% and 97% by single step, respectively. And the reporter gene experiment of the target protein activating Notch signal results indicate that the purified rhDlll is biologically active with dose-dependent activation of Notch signal. |
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