酵母双杂交系统ppt课件.ppt
酵母双杂交系统,扬州大学生物科学与技术学院,酵母双杂交系统应用,鉴定新的蛋白与蛋白相互作用鉴定蛋白级联底物 鉴定突变对蛋白与蛋白结合的影响在已知的相互作用中鉴定干扰蛋白质 (反向双杂交系统),酵母双杂交的原理,利用酵母作为真核蛋白相互作用的模型利用诱饵质粒筛选文库或者单个已知蛋白通过报告基因的转录鉴定蛋白的相互作用使用不同的培养基筛选阳性克隆,酵母双杂交模型,DNA-BindingDomain,Bait Protein,Prey Protein,TranscriptionActivatingRegion,Reporter Gene,DNA-Binding Site,模型,文库筛选的步骤,将待测基因与Gal4或LexA或其他合适蛋白的DNA结合域融合构建诱饵质粒将诱饵质粒转化缺乏报告基因启动子的酵母细胞株中，选择被转化的酵母再将文库质粒转化到酵母中通过报告基因的功能筛选相互作用的蛋白,序列分析,从酵母中分离质粒然后转化E. coli从大肠杆菌中提取质粒并测序在数据库中比对所测定序列与已知蛋白的同源性,报告基因,LacZ reporter - Blue/White ScreeningHIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity)LEU2 reporter - Screen on Leu+ mediaADE2 reporter - Screen on Ade+ mediaURA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA),质粒构建,在Gal4（或其他合适的蛋白功能域）的 DNA结合域前面的多克隆位点处插入待测基因序列构建诱饵质粒诱饵质粒含有筛选基因，例如氨苄青霉素抗性基因，Leu2, Ura3, or Trp1等。,质粒样品,From Golemis Lab Homepage,假阳性的问题,假阳性是酵母双杂交系统的最大问题假阳性通常由以下原因造成:prey蛋白的非特异性结合无需与诱饵蛋白结合就能诱导报告基因的转录（这是大多数假阳性的诱因）,假阳性的去除,通过序列分析去除质粒消除实验剔除假阳性重新转化含有诱饵质粒的酵母株或不含诱饵质粒的酵母株，分析两种情况后去除假阳性利用一个不相关的蛋白作为诱饵蛋白测试相互作用两不或多步筛选,酵母双杂交的优点,快速获得相互作用蛋白的基因只需单一步骤的质粒构建在体内鉴定蛋白的相互作用弱小的，暂时的相互作用也能鉴定能在整个时间段积聚弱小的相互作用信号,酵母双杂交应用举例,蛋白级联底物的鉴定钙调蛋白与L-异天冬氨酰甲基转移酶的相互作用E2F1 突变子的遗传特性多肽荷尔蒙受体相互作用Pha-4在线虫 C. elegans 中 的相互作用,参考文献,Bartel, Paul, C. Chien, R. Sternglanz, S. Fields. “Elimination of False Positives that Arise in Using the Two-Hybrid System.” Biotechniques (1993) Vol. 14, no. 6, p. 920-924.Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. “The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest.” Proc. Natl. Acad. Sci. USA (1991) Vol. 88, p. 9578-9582.Fields, Stanley, O. Song. A novel genetic system to detect protein-protein interactions. Nature (1989) Vol. 340, p.245-246.James, Philip, J. Halladay, E. Craig. Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast. Genetics (1996) Vol. 144, p. 1425-1436.Kamada, S, H. Kusano, H. Fujita, M. Ohtsu, R. Koya, N. Kuzumaki, Y. Tsujimoto. A cloning method for caspase substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin. Proc. Natl. Acad. Sci. USA (1998) Vol 95, p. 8532-8537.OConnor, Mirriam, C. OConnor. Complex Interactions of the Protein L-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System. The Journal of Biological Chemistry (1998) Vol. 273, p. 12909-12913.Staudinger, Jeff, J. Zhou, R. Burgess, S. Elledge, E. Olson. PICK1: A Perinuclear Binding Protein and Substrate for Protein Kinase C Isolated by the Yeast Two-Hybrid System. The Journal of Cell Biology (1995) Vol. 128, p. 263-271.,References continued,Vidal, Marc, P. Braun, E. Chen, J. Boeke, E. Harlow. Genetic Characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proc. Natl. Acad. Sci. USA (1996) Vol. 93, p. 10321-10326.White, Michael. The yeast two-hybrid system: Forward and reverse. Proc. Natl. Acad. Sci. USA (1996) Vol 93, p. 10001-10003.Zhu, Jianwei, C. Kahn. Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system. Proc. Natl. Acad. Sci. USA (1997) Vol. 94, p. 13063-13068.Lab of Erica Golemis http:/www.fccc.edu/research/labs/golemis/EG_homepage.htmlSpecial thanks to Dr. Susan Mango and the University of Utah,
