Research On Renaturation And Purification Of Pharmaceutical Protein Expressed As Inclusion Body In Recombinant E.Coli

E.coli expression system is still the most widely used recombinant protein expression systems.On account of the fast expression or incorrect match of disulfide bond,the heterologous proteins are often expressed in the form of inactive inclusion bodies in E.coli.As the folding mechanism of inclusion complex is not clear,it is so difficult to refold the inactive inclusion body to completely natural active state.Low refolding efficiency is still the main reason of low yield,or hampering the preparation of exogenous proteins,when E.coli is used as expression system.In this study,we selected recombinant human epidermal growth factor(hEGF)and peptide-based HIV vaccine as the research objects to study how to deal with the order between refolding and purification,how to select methods of refolding and purification,how to design a reasonable scheme of purification(link up with different purification methods as well as possible),based on specific requirements and purposes,which will provide some reference for other pharmaceutical proteins expressed in the form of inclusion body.Renaturation and purification of non-fusion hEGF.The strategy of "refolding followed by purification" was applied in this study.We developed the process of refolding and purification of hEGF as follows:Washing of the inclusion bodies ? Denaturation and renaturation of the inclusion bodies?(50%saturation)Ammonium sulfate precipitation?Gel filtration chromatography ? DEAE anion exchange chromatography.SDS-PAGE and HPLC analysis showed the purity of the purified hEGF was 97%and 96%respectively.The protein recovery rate of hEGF was 18%.And this purified hEGF showed a significant activity to promote the growth of the Balb/c 3T3 cells(mouse embryonic fibroblasts).Renaturation and purification of a novel pharmaceutical protein-HIV T helper vaccine(fusion expression).(1)Optimization of denaturation conditions.The Inclusion bodies was first dissolved in denaturation buffer containing 7 M guanidine-HCl).Then the supernatant was diluted by 8 M urea buffer to decrease the concentration of guanidine-HCl to 1 M.Thus,the protein structure of denatured inclusion bodies tended to be more stable.And no protein precipitation appeared in the process of purification and refolding.(2)Purification and refolding of HIV vaccine inclusion bodies.The scheme of "purification followed by refolding" was selected to use in this section.We developed the technics of refolding and purification of HIV vaccine as follows:Washing of the inclusion bodies? Denaturation of inclusion bodies? Nickel affinity chromatography(HisTrap FF)?(30%saturation)Ammonium sulfate precipitation ? CM cation exchange chromatography ? Nickel affinity chromatography(HisTrap HP).Final purity of recombinant protein was up to 95%.And the purification recovery rate of HIV vaccine was 13%.Purified inclusion-body protein with denatured state was renatured by stepwise dialysis without obvious precipitation.Purification and refolding of HIV vaccine provided the guarantee for further research on physico-chemical properties and biological function of HIV T helper vaccine.