蛋白质结晶学和X光衍射基础课件.ppt

* 每一项革命性技术的产生都将推动众多领域的跨越式发展；是美国科学家约翰·芬恩与日本科学家田中耕一“发明了对生物大分子的质谱分析法”，他们两人将共享2002年诺贝尔化学奖一半的奖金； * 宝石晶体(a)和硅单晶体 * * 香港和美国科学家联合宣布，他们找到了一种能够有效抑制流感病毒、包括H5N1禽流感. * To study the crystal structure of a protein or protein complex, enough highly purified protein to crystallize is the vital step. Since different vectors, different tags, different bacteria strains or different eukaryotic host cells may influence the expression and behavior of the recombinant protein, to obtain a recombinant protein, sometimes I have to try different ways one by one. No try, no confirm. The following 5 slides show some different vectors used for expressing my target proteins in varies host cells. * This is one of easiest yeast expression system vector. The cloned gene will recombinant to yeast chromosome and the expression is induced by methanol. * This is a easy-handling vector for insect cell expression. The advantage of this vector system is: before the virus infects the cells, it has been confirmed recombinant, so the recombinant efficiency would be 100% theoretically in insect cell. Bacmid是一种杆状病毒穿梭载体(Baculovirus　shuttle　 vector) * The human 293 cell line is widely used for r-protein production as it offers many advantages such as high transfection yields with most gene transfer vehicles, is easily grown in suspension culture, and can be adapted to serum-free media. Moreover, two genetic variants, the 293E and 293T cell lines, expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication. Thus, they are expected to increase r-protein expression levels by permitting more plasmid copies to persist in the transfected cells throughout the production phase. Since the cheap reagent PEI（聚醚酰亚胺） is developed for mammalian cell transfection, it made large scale suspension cell transfection more feasible. * These two figures show the transfection efficienc