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酵母双杂交系统

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. Transcription Activating Region. Bait Protein. Prey Protein. DNA-Binding Domain.

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酵母双杂交系统

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  1. 酵母双杂交系统 扬州大学生物科学与技术学院

  2. 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)

  3. 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

  4. 酵母双杂交模型 Transcription Activating Region Bait Protein Prey Protein DNA-Binding Domain Reporter Gene DNA-Binding Site

  5. 模型

  6. 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

  7. 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

  8. 报告基因 • LacZ reporter - Blue/White Screening • HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity) • LEU2 reporter - Screen on Leu+ media • ADE2 reporter - Screen on Ade+ media • URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)

  9. 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。

  10. 质粒样品 From Golemis Lab Homepage

  11. 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）

  12. 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

  13. 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

  14. 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

  15. 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. • O'Connor, Mirriam, C. O'Connor. "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.

  16. 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.html • Special thanks to Dr. Susan Mango and the University of Utah

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