Study Of HAb18G/CD147 Extracellular Portion Protein Refolding

HAb18G/CD147 is a widely expressed transmembrane glycoprotein and has been involved in many physiological and pathological processes, especially in tumor invasion and metastasis, and inflammation development. But the exact molecular mechanism of the functions is far from fully understood and the three-dimensional structure of HAb18G/CD147 remains unknown. The extracellular portion of HAb18G/CD147 molecule (HAb18GEP) has been identified to be responsible for most of the functions. It is necessary to determine the three-dimensional structure of HAb18GEP, for which, preparing plenty of HAb18GEP protein with high purity and homogeneity becomes to the primary precondition and the important basis.Considering the inherent limitation of eukaryotic expression system, prokaryotic expression system is commonly used to produce exogenous proteins for structure study. Nevertheless, exogenous proteins often form inactive inclusion body when expressed in the prokaryotic system, which is the main baffle in protein preparation and structure study. On the other hand,how protein folding to native structure has puzzled scientists for a long time. Studies on folding pathway and refolding mechanism will contribute to clarify the relationship between protein structure and protein function, and thus lay a profound significance for exploring the essence of life. Based on all the above, the present study aims to establish a high efficient refolding method to prepare HAb18GEP protein in native structure with high purity and homogeneity for subsequent structure research. The second aim is to preliminarily explore the HAb18GEP refolding intermediate according to the"folding pathway theory".The present study consists of four parts:1. Prokaryotic expression and purification of HAb18GEP protein.After screening appropriate stains and optimizing culture conditions, we successfully produced deglycosylated HAb18GEP with high expression efficiency in Escherichia coli. The protein product was mainly in the form of inclusion body and a little in the soluble form. According to their different structural characteristics, the inclusion body and soluble protein of HAb18GEP was respectively purified by hydrophobic interaction chromatography (HIC) and anion-exchange chromatography (IEC) with a purity of over 95%. Analyzed with ELISA, the two forms of HAb18GEP protein were found to be both able to bind to HAb18G/CD147 specific monoclonal antibody (mAb), but the affinity of inclusion body to the mAb was lower that that of soluble protein.The preparation of HAb18GEP inclusion body with high purity lays a basis of high-efficiency refolding. The purified soluble HAb18GEP can be used as the standard native protein for refolding study. 2.HAb18GEP refolding with glycerol-mediated HIC.A novel strategy combining glycerol and HIC media was introduced to facilitate HAb18GEP refolding. We examined the effects of medium, glycerol and other relative factors on refolding rate and compared this strategy with the dilution refolding method.Our results proved the previous findings that the commercial hydrophobic chromatographic media could inhibit the protein aggregation in the refolding process, which was negatively related to the hydrophobicity of the media, but could lead to the irreversible adsorption of proteins to the media. The addition of glycerol to the mobile phase could prevent the irreversible adsorption and facilitate the correct refolding of protein. Then we designed and developed a new strategy to refold HAb18GEP with 20%-glycerol-mediated Poros ET HIC and successfully refolded HAb18GEP with a soluble protein recovery rate of 83% and a correct refolding rate of 30%. These results suggest that combining weak hydrophobic interaction media with glycerol elution in appropriate concentration can not only inhibit protein coagulation, but also promote the correct refolding of protein.3.HAb18GEP refolding with double-gradient IEC.Using traditional dilution process, we examined the effects of protein concentration, temperature, salts and additives on the refolding efficiency of HAb18GEP. We then explored the effects of the above factors on HAb18GEP refolding with IEC.Our results showed that the pH value of refolding buffer was an important factor for the correct refolding of protein and the correct pairing of disulfide bonds. As HAb18GEP comprising two pairs of disulfide bonds, we introduced the pH gradient into IEC for HAb18GEP refolding. After denatured protein was adsorbed on the chromatography media, the concentration of the denaturant was gradually decreased while the pH value was gradually increased, which facilitate the denatured protein was correctly refolded step by step.With this method, HAb18GEP was successfully refolded with a soluble protein recovery rate of 95% and a correct refolding rate of 67%, which exhibited a higher efficiency than that with glycerol-mediated HIC.After separation with reversion phase chromatography, the correctly refolded protein was analyzed for its bioactivity with ELISA and conformation with circular dichroism spectrum (CD), intrinsic fluorescence spectrum and nuclear magnetic resonance (NMR). The results showed that the refolded HAb18GEP was well-structured at both the secondary and tertiary structure levels and retained the same physico-chemical property with the native protein.4.Study on the equilibrium molten-globule intermediate of HAb18GEP.Protein function is closely related to its three-dimensional structure. According to the"folding pathway theory", the formation of intermediate is a key step in protein folding to reduce the searching scope for protein refolding. In the present study, HAb18GEP induced by GdnHCl leads to a three-state unfolding process with the formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of GdnHCl. On the coutary, urea denaturation is found to be a simple two-state transition. Inclusion of 1.5M NaCl to the urea denaturant to mimic the ionic character of GdnHCl leads to a three-state unfolding behavior with a significantly stabilized molten-globule intermediate by the chloride salt. We thus presumed that there might be some hidden intermediates in the folding pathway of HAb18GEP.In summary, the present study successfully refolds HAb18GEP inclusion body respectively with HIC and IEC, whereas IEC with a higher refolding efficiency. The structure and bioactivity of the refolded protein are consistent with those of the deglycosylated native HAb18GEP. Now the refolded HAb18GEP has been used in the detection and analysis of HAb18GEP structure with NMR. The methods established in the present study provide experiences and clues for the refolding of other similar transmembrane proteins.Furthermore, based on the"folding pathway theory", we find that NaCl can induce urea-denatured HAb18GEP to form unfolding molten-globule intermediate, which will provide experiment evidence for further investigation of protein refolding pathway and promote the understanding of different refolding theories.