蛋白质结晶学和X光衍射基础幻灯片.ppt

Thank you for your attention * 人类基因组图谱、水稻基因组草图以及其他一些生物基因组图谱破译成功后，生命科学和生物技术进入后基因组时代。这一时代的重点课题是破译基因的功能，破译蛋白质的结构和功能，破译基因怎样控制合成蛋白质，蛋白质又是怎样发挥生理作用等。在这些课题中，判定生物大分子的身份，“看清”它们的结构非常重要。 瑞士科学家库尔特·维特里希“发明了利用核磁共振技术测定溶液中生物大分子三维结构的方法”，他将获得2002年诺贝尔化学奖一半的奖金。 * 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 trans