| The rapid development of genetic engineering makes large-scale production of recombinant protein as possible, but during the industrial drug development, two critical "bottleneck" problems have to be solved. First, the over-expression of heterologous protein in microorganisms such as Escherichia coli often results in the formation of intracellular insoluble protein, also known as inclusion bodies. These inclusion bodies must be refolded in vitro to gain solubility and bioactivity. However, the experiential methods are still prevalent in the research of protein refolding at home and abroad, which have little value in promotion of industrialization. The refolding additives have been reported to suppress aggregation by preventing the association of refolding intermediates or unfolded species through hydrophobic or ionic interactions. But the interaction of proteins with the vast majority of refolding additives is irreversible, which makes follow-up protein separation and purification have many difficulties. Second, in the practical application of the process, the correct folding of functional proteins has the defect such as poor stability and short half-life in vivo. This makes protein drugs difficulty to conserve, high frequency use and high cost of medical treatment, which restrict severely the protein drugs industrialization and clinical application.To address the above-mentioned "bottleneck" problem, this paper carried out two studies: First, a new type of refolded additive was studied on an intelligent poly-compound Eudragit S-100:whether it can promote protein refolding, obtain its role of advantages compared with existing methods. On this basis, the mechanisms of Eudragit-assisted protein refolding were investigated, specifically including:1. It was studied whether Eudragit S-100 can promote protein refolding from the activity, content and structure of refolded protein, with lysozyme as a model protein. This study showed that (ⅰ) the addition of Eudragit S-100 in the refolding buffer significantly increased lysozyme refolding yield to 81%, when the concentration of it was up to 0.4%(w/v) and dilution refolding was conducted at 1 mg/mL lysozyme; (ⅱ) Eudragit-lysozyme interaction did not compromise the refolded protein conformation, as confirmed by fluorescence and circular dichroism spectroscopy.2. According to the physical and chemical properties of Eudragit S-100. precipitation and ion-exchange experiments showed the evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit S-100 polymer during refolding. These ionic complexes of Eudragit S-100 and lysozyme appeared to shield expose hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic-driven aggregation of the molecules. Importantly, due to the electrostatic interaction is reversible; the polymer can be readily dissociated from the protein by ion exchange chromatography.3. Through the molecular mechanism of Eudragit S-100 promoting protein refolding, we found an interesting speculation that Eudragit S-100 can only promote the refolding of basic proteins. In order to verify the correctness of this speculation, we selected two ionic forms of human fibroblast growth factor:(ⅰ) human basic fibroblast growth factor (hbFGF) and (ii) human acidic fibroblast growth factor (haFGF) as model proteins. The results showed that Eudragit S-100 only enhanced the refolding yield of hbFGF, but little effect on haFGF. Finally, we successfully applied this strategy to refold another two basic proteins KGF-2 (keratinocyte growth factor-2) and TGF-β1 (transforming growth factor-(31).The second part focused on the study of PEG-phenyl-isothiocyanate enhancing protein stability studies, specifically including:1. In present study, we modified keratinocyte growth factor-2(KGF-2) by PEGylation at the N-terminal residue using the above-mentioned PEGylation reagent. The effects of reactants molar ratio, temperature and pH on the PEGylation were examined through the three-factor three-level orthogonal experiments. The optimized modification conditions ultimately determined:the molar ratio of PIT-PEG20K to KGF-2 was 9:1, the reaction temperature was 25℃, pH value was 6.0. Under the modified conditions, KGF-2 production rate of the modified was 31.63%. Under this condition, the yield of PEGylated rhKGF-2 reached a significant amount of 37.8%. PEGylated KGF-2 was then purified by a Heparin Sepharose TM CL-6B affinity chromatography and its purity was over 99%.2. The biological properties of PEGylated KGF-2 were systematically studied. The results showed that the PEGylated rhKGF-2 retained about 60% of mitogenic activity compared with the non-modified rhKGF-2. Its relative thermal stability at normal physiological temperature and structural stability were significantly enhanced. Spatial structure of protein had no significant change. The half-life time in vivo prolonged from 2.6 min to 15.5 min. Moreover, the immunogenicity of PEGylated rhKGF-2 in mice decreased significantly as compared with non-modified rhKGF-2.In conclusion, we found an efficient refolding method by the above-mentioned studies for basic proteins. Furthermore, we also successfully made a more stable KGF-2 by PEG-modified method. These studies provide some good ideas and tools to solve two critical "bottleneck" problems of protein engineering.
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