Purification And Characterization Of Non-taged Recombinant Human Cytoglobin

Cytoglobin(Cygb)was first discovered in 2001 by proteomic analysis in rat stellate cells and was first named Stellate Activating Protein(STAP).Cygb consists of 190 amino acids with acalculated molecular mass of 21 kDa.Cygb is a hexacoordinated heme-containing globin.As a member of the globin family,which includes myoglobin,hemoglobin,and neuroglobin,Cygb has the same function of binding and transporting oxygen.And Cygb facilitates oxygen(O2)diffusion through tissues,scavenges nitric oxide(NO)and other ROS.It’s reported that Cygb plays an important role in controlling tissue fibrosis,such as liver,kidney,and pancreas and glaucoma,gastric tube reflux and some inflammation,neurodegenerative diseases etc.Cygb has numerous biological functions.Our previous studys showed that recombinant human cytoglobin can reverse the formation and development of Liver fibrosis,pulmonary fibrosis,fatty liver and hyperlipidemia and atherosclerosis effectively etc.This study was based on the efficacy of the above-mentioned conditions in our laboratory,with the aim of providing Cygb samples that meet the purity criteria next clinical trials.The preparation of rhCygb was induced by BL21/pET28a(+)-rhCygb,which was expressed in the form of unlabeled inclusion bodies.The purification process needed to be improved and optimized.We used three methods,namely,immunoaffinity chromatography,anion exchange chromatography and gel filtration chromatography.Q Sepharose Fast Flow chromatography combined with Sephacryl-100 chromatography were used to obtained high purity rhCygb.And we studied the rhCygb biological activity of antioxidant and antiinflammatory in vitro.This study is divided into four parts.The first part is the preparation of rhCygb,and the inclusion bodies which do not have biological activity are obtained.We discuss the different methods of washing inclusions and refolding inclusion bodies.The experimental results showed that we got the ideal inclusion bodies with Sephadex G-25.The second part is purifying rhCygb with immunoaffinity chromatography.The anti-rhCygb mice ascites of monoclonal antibody were purified by proteinA.And the specificity and affinity of the monoclonal antibody were the key factor in the antibody affinity chromatography.A sample was taken for analyses on SDS-PAGE,and the results showed that the purity of rhCygb product was less than 90%,and the protein was enriched by ultrafiltration tube with white flocculent.Considering the extreme conditions of affinity chromatography elution,the cyanide and antibody are easy to fall off from Sepharose4B.Although immunoaffinity chromatography purification of purified protein is broader,usually as the first choice for protein purification,However,this method is not suitable for the purification of rhCygb.The third part is anion exchange and gel filtration chromatography to purify rhCygb.We used Q-Sepharose to obtain the rhCygb of 89.34%purity,and further using gel filtration chromatography Sephacryl-100 to separate different molecular weight between rhCygb and other proteins.And this method achieves the purpose with 92.6336%purity determined by HPLC.And Western blot was used to identify the immunological activity.The fourth part is analysis of the biological activity of rhCygb,which mainly studied the biological activity of rhCygb in anti-inflammatory and anti-oxidation.The antioxidant capacity in vitro was determined by detecting DPPH.BV2 mouse microglia was induced by Oxygen-glucosedeprivation(OGD)to simulate the cell of inflammation model in vivo.After oxygen-glucose deprivation recovery(OGDR)and rhCygb intervention to the above model,we studied the effect of rhCygb on the cell activity and ROS content of the cell.The results showed that rhCygb has protective effect on the cell of inflammation model and the decrease of ROS content.Conclusion:1.The expression and purification method of Non-taged rhCygb was established.And obtaind the purity of 92.6336%rhCygb to meet the purity criteria next clinical trials.2.Demonstrates that the effect of rhCygb on antioxidant activity and anti-inflammatory.