Effect Of Small Molecule Additives On Protein Refolding In Vitro And Its Process Analysis

By far, adding small molecule reagents in solution is still one of the strategies to improve the protein refolding efficiency in vitro. Its purpose is to reduce protein aggregation and protein precipitattion as well as to promote conformation change of proteins from denatured states to native and active states during refolding. To a certain extent, this method can improve the efficiency of protein refolding and has been developed quite well. In this paper, in order to capture the changing regulation of difference from the structure of denatured protein to the active, and to definite the mechanisms of small molecule additives on protein refolding in vitro, lysozyme was used as a model standard protein and rhFL expressed in E.coli was used as a model protein from inclusion bodies to study the effects of three kinds of small molecule additives on the proteins refolding by using different renaturation methods and fluorescence spectroscopy. The relationship between the change of proteins conformation and protein refolding efficiency, fluorescence intensity in refolding process was also investigated. The obtained results provided lots of theoretical knowledge in the protein conformational change and reliable data and guidance for solving the problem that it is difficult to refold proteins from inclusion bodies and their refolding efficiency is low.1. Literature review:The recent advances in protein refolding, including pretreatment of proteins, methods for protein refolding in vitro and effect of different small molecule additives on protein refolding in vitro were reviewed.2. Refolding of lysozyme by dilution and characterization of its conformation by fluorescence:In this study, the native lysozyme was took as a standard protein model to investigate the effect of different small molecule additives on the protein activity recovery and fluorescence spectroscopy during refolding by dilution, the mechanism of small molecules on protein refolding, and the relationship between the determination results were analyzed.3. Refolding of lysozyme by HPHIC and characterization of its conformation by fluorescence:The denatured Lys was applied as a target protein in this study, different concentrations of urea, glutathione (GSH/GSSG), L-arginine were added to refolding process by high performance hydrophobic interaction chromatography (HPHIC). Protein mass recovery, activity recovery and fluorescence spectra were investigated before or after refolding, and the influence of small molecule additives on refolding of denatured Lys and its mechanism were analysed. The results showed that these small molecular additives can improve efficiency of protein refolding to different extent. Product of refolding was further identified and analysed, it was found that Lys refolding results including refolding efficiency, fluorescence spectra and conformational change by IIPIIIC were consistent with the results by dilution.4. Refolding and simultaneous purification of rhFL by HPHIC:E. coli cells containing FL inclusion bodies were collected by centrifugation and the cells were disrupted by sonication. After washing several times, the purified inclusion bodies were dissolved in 8 mol/L urea. Urea, glutathione (GSH/GSSG) and L-arginine were added in HPHIC mobile phase to study the effect of different kinds and concentrations of small molecules additives on renaturation and simultaneous purification of rhFL. The fluorescence intensity of the target protein before or after refolding and refolding effect was further studied by fluorescence spectroscopy. The results showed that the mass recovery of rhFL can reach 38.4% with 3 mol/L urea,1.8 mmol/L/0.3 mmol/L (GSH/GSSG) and 0.5 mol/L L-arginine in the mobile phases, it's easy to restore the biological activity in such condition. The results obtained for rhFL lay a foundation for the development of rhFL drugs.