Study On Inclusion Body Protein Refolding Assisted By Hydrophobic Interaction Chromatography And Osmolytes

In-vitro inclusion body protein refolding is one of bottlenecks in downstream process of biotechnology. Usually biologically active proteins can not be obtained with high activity yield at a high concentration by conventional dilution refolding method because of arise a large of misfolding and aggregation during renaturation. The objective of this research is to develop a hydrophobic interaction chromatography assisting protein renaturation method in order to meet the challenge.The dissertation is divided into four parts. The first part focused on optimizing dilution refolding conditions and dilution refolding ways of consensus interferon (C-IFN). As an additive of dilution refolding buffer, the effects of polyethylene glycol (PEG), glycerol and some media such as: Butyl FF, Phenyl FF, Poros ET and Poros PE on the activity and yield of protein refolded by dilution refolding was discussed. The refolding buffer of Poros ET medium added 10 % PEG was found to be most suitable in dilution refolding of C-IFN. The protein yield was 74.6% and the specific activity was 2.61 ×10~8 U / mg, however about 45.4 % protein yield with 2.1 ×10~8 U / mg specific activity was obtained with traditional refolding buffer under the same dilution method.The second part focused on analysis of different configurations of C-IFN refolded in dilution by a series of methods which were reverse-phase-HPLC, gel filtration chromatography, ion-exchange chromatography, non-reduced / reduced SDS-PAGE analysis and mass spectrometric assignment of disulfide bonds. A new model of reverse-phase-HPLC was developed to distinguish misfolding conformations of C-IFN, soluble aggregation of C-IFN and correct configurations of C-IFN.The third part focused on study on hydrophobic interaction chromatography assisting C-IFN renaturation. It was discovered that commercially available hydrophobic interaction chromatographic media, which are used for protein purification, were not able to assist protein refolding if being employed directly in the same way as in protein purification. Low activity yield and irreversible adsorption were found. However, this problem was partly circumvented by addition of PEG to the mobile phase of the chromatography and utilization a short time elution of high concentration guanidine-HCl. But, there was still a large of misfolding configurations of C-IFN in the refolded mixture and the concentration of refolded protein was not high. A novel dual-gradient hydrophobic interaction chromatography, consisting of decreasing guanidine-HCl concentration and increasing PEG concentration, was developed based on the principle of hydrophobic interaction chromatography. Which...