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The Prokaryotic Expression and Renaturation of CD147 Extracellular Portion, and the Preliminary Research of Solution Structure

Author: RuQiang
Tutor: ChenZhiNan；SongZuo
School: Fourth Military Medical University
Course: Cell Biology
Keywords: HAb18GEP NMR Inclusion bodies Protein Folding Protein refolding
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 69
Quote: 0
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Abstract


HAb18G/CD147 belonging to a member of the immunoglobulin superfamily, is widely expressed in the body a single transmembrane glycoprotein, involved in many physiological processes in the life activities, such as tumor cell invasion and metastasis, the migration of lymphocytes and activation, tissue repair and remodeling, inflammation, Alzheimer's disease and other, and plays an important biological function. And many of the features of the play depends on it extracellular segment (HAb18GEP) of its glycosylation. Therefore, the study of the spatial structure of HAb18GEP, so as to clarify the mechanism of the interaction with other molecules, and the understanding of how it functions is very necessary. The main advantage of NMR techniques given level atomic resolution structure of biological macromolecules in solution, conformation, in particular, can be quickly and easily to study protein-protein interactions, to obtain biological macromolecules in the actual situation of the organism. Therefore, we have adopted the NMR method of The structure of HAb18GEP preliminary study. Because HAb18GEP protein expression in prokaryotic systems exist mainly in the form of inclusion bodies, this study explored a set for HAb18GEP effective in vitro refolding method, and its renaturation. Preliminary study of the structure of HAb18GEP its two domains using nuclear magnetic resonance technology, the completion of the identification of the main chain of C80, the solution structure and protein interactions has laid a good foundation for the next step. This study is divided into three parts: (a) HAb18GEP expression in Escherichia coli, renaturation and stability studies to optimize culture conditions to achieve efficient expression of the HAb18GEP get a lot of protein in the form of inclusion bodies, and the success of the vitro protein refolding. The application of NMR technology proved good folding protein. The protein stability in the study to confirm the protein degradation product 9.5kDa peptide fragment and the protein sample solution conditions were optimized to improve the quality of the data of the NMR experiments. (B) HAb18GEP C-domain and the expression of N-domain, renaturation according HAb18GEP secondary structure prediction and its stability studies, respectively, the two domains HAb18GEP cloning, expression. On the N90 and C80 protein expression, in vitro refolding, purification, NMR experiments prove good folding. (C) the main chain of the C80 protein refers recognized on the C80 protein isotope labeling, expression, in vitro refolding and purification, NMR experiments prove having good folding. We collect C80 All main chain resonance data and its analysis, 85% of the main chain CHN atoms the chemical shifts of vesting. The work for further solution structure research laid the foundation, and provide the basis for subsequent protein interaction studies.

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