The Prokaryotic Expression And Renaturation Of CD147 Extracellular Portion, And The Preliminary Research Of Solution Structure

HAb18G/CD147 is a member of the immunoglobulin superfamily. It is a widely expressed transmembrane glycoprotein in organism and has been involved in many physiological and pathological processes, such as the invasion and metastasis of tumor cell, lymphocyte migration and activation, tissue repair and remodeling, inflammation and Alzheimer's disease. Most of its functions are depended on its extracellular domains and its glycosylation. Therefore, the investigation of the 3D structure of the HAb18GEP is very important for the elucidation of its function mechanism.The major advantage of NMR technique is that it can give the structure and conformation information of biomacromolecule in solution, especially for the convenient and fast research of protein-protein interaction, therefore gains the actuality condition of the biomacromolecule in the organism.Therefore, we use the NMR method to do the preliminary research of the structure of HAb18GEP. Since HAb18GEP protein expressed in the prokaryotic expression system is in inclusion body form, our studies developed an effective renaturation method for the HAb18GEP, and successfully achieved the renaturation of HAb18GEP. Preliminary characterization of full length HAb18GEP and its two domains with NMR showed that they are all well folded. The assignments of backbone atoms of the diomain C80 were completed for 85%. The studies provide the foundation for the future research of solution structure and protein-protein interaction.The present study consists of three parts:1: The expression of HAb18GEP in E coli, the renaturation of HAb18GEP, and the stability investigationBy optimizing the culture condition, HAb18GEP was high level expressed. A great quantity of protein was obtained in the form of inclusion body. The renaturation of protein was successfully accomplished. After the folding of protein was analyzed with NMR, the protein was confirmed that it is well folded. According to the result of the protein stability study, the product of protein degradation was confirmed that it is a 9.5kDa peptide. By optimizing the solution condition for the storage of HAb18GEP, the quality of the NMR experimental data was improved.2: The expression and renaturation of the C-Domain and N-Domain of HAb18GEPAccording to the secondary structure prediction and stability research of HAb18GEP, the C-Domain and N-Domain of HAb18GEP were cloned and expressed. N90 and C80 were selected to express, renature, and purify. After the folding of proteins were analyzed with NMR, the proteins were conformed that they are well folded. 3: Backbone NMR signal assignments of C80C80 was labeled with the isotope, expressed in the prokaryotic expression system, renatured in vitro, and purified. After the folding of protein was analyzed with NMR, the protein was confirmed that it is also well folded. We collected triple-resonance experiments data of all C80 backbone, and analyzed them. The assignments of backbone atoms C, H, and N of the domain C80 were completed for 85%. The studies provide the foundation for the future research of solution structure and protein-protein interaction.