分子大实验乃哥麦提.docx

Comprehensive experiments of Biochemistry and Molecular Biology Molecular Part 生13 杨林枫 2011012326 同组人员：乃哥麦提 褚培睿 I. Objectives 1. Learn to design an entire molecular biology experiment to get a system which can express protein. 2. Review the molecular biology experiment technology we learned before. 3. Prepare the samples for part II and part III. 4. Organizing each assay to get fully use of the time. 5. Corporate with others. II. Background HSP16.3 HSP16.3, a small Heat Shock Protein from M.tuberculosis, consists of 144 amino acids and has a molecular weight of 16277 Da. It is significantly expressed when M.tuberculosis changes from logarithm phase to stable phase and becomes main protein in the cell. HSP16.3 is also an antigen, important target site of T and B cell in immune response. Experiments in vitro indicate that HSP16.3 has a chaperone activity, and it can inhibit thermal aggregation of citric acid synthetase at 39.5 QUOTE without consumption of ATP. But it cannot protect citric acid synthetase activity, which indicates that HSP16.3 interacts with partially unfolding citric acid synthetase and refolding of protein in vivo may need other proteins. Gel chromatography can separate HSP16.3-citric acid synthetase complex, and SDScan identify them. HSP16.3 may expose its hydrophobic surface to carry out its chaperone activity. With mild temperature and mutagen in low concentration, for example, under the condition of 0.05 M Guanidinium chloride, 0.3 M urea and 30 QUOTE , HSP16.3 will expose its hydrophobic surface and elevate its chaperone activity. HSP16.3 is a member of QUOTE -crystalline associated small Heat Shock Protein (sHSP) family, with the typical conserved CTD: D/N-G-V-L-T-T/V-X-V/A. sHSP have many different biological functions. They all have chaperone activity, interacting with denatured protein substrate even without ATP. Research shows that they often form certain oligomer structure to carry out its function, for example, M.Janneschii HSP16.5 f