酵母雙雜交系統(tǒng)揚(yáng)州大學(xué)

1、酵母雙雜交系統(tǒng)揚(yáng)州大學(xué)生物科學(xué)與技術(shù)學(xué)院酵母雙雜交系統(tǒng)應(yīng)用酵母雙雜交系統(tǒng)應(yīng)用 鑒定新的蛋白與蛋白相互作用 鑒定蛋白級(jí)聯(lián)底物 鑒定突變對(duì)蛋白與蛋白結(jié)合的影響 在已知的相互作用中鑒定干擾蛋白質(zhì) (反向雙雜交系統(tǒng))酵母雙雜交的原理 利用酵母作為真核蛋白相互作用的模型 利用誘餌質(zhì)粒篩選文庫(kù)或者單個(gè)已知蛋白 通過(guò)報(bào)告基因的轉(zhuǎn)錄鑒定蛋白的相互作用 使用不同的培養(yǎng)基篩選陽(yáng)性克隆酵母雙雜交模型DNA-BindingDomainBait ProteinPrey ProteinTranscriptionActivatingRegionReporter GeneDNA-Binding Site模型文庫(kù)篩選的步驟 將

2、待測(cè)基因與Gal4或LexA或其他合適蛋白的DNA結(jié)合域融合構(gòu)建誘餌質(zhì)粒 將誘餌質(zhì)粒轉(zhuǎn)化缺乏報(bào)告基因啟動(dòng)子的酵母細(xì)胞株中，選擇被轉(zhuǎn)化的酵母 再將文庫(kù)質(zhì)粒轉(zhuǎn)化到酵母中 通過(guò)報(bào)告基因的功能篩選相互作用的蛋白序列分析 從酵母中分離質(zhì)粒然后轉(zhuǎn)化E. coli 從大腸桿菌中提取質(zhì)粒并測(cè)序 在數(shù)據(jù)庫(kù)中比對(duì)所測(cè)定序列與已知蛋白的同源性報(bào)告基因 LacZ reporter - Blue/White Screening HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity) LEU2 repor

3、ter - Screen on Leu+ media ADE2 reporter - Screen on Ade+ media URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)質(zhì)粒構(gòu)建 在Gal4（或其他合適的蛋白功能域）的 DNA結(jié)合域前面的多克隆位點(diǎn)處插入待測(cè)基因序列構(gòu)建誘餌質(zhì)粒 誘餌質(zhì)粒含有篩選基因，例如氨芐青霉素抗性基因，Leu2, Ura3, or Trp1等。質(zhì)粒樣品From Golemis Lab Homepage假陽(yáng)性的問(wèn)題假陽(yáng)性是酵母雙雜交系統(tǒng)的最大問(wèn)題假陽(yáng)性通常由以下

4、原因造成:1. prey蛋白的非特異性結(jié)合2. 無(wú)需與誘餌蛋白結(jié)合就能誘導(dǎo)報(bào)告基因的轉(zhuǎn)錄（這是大多數(shù)假陽(yáng)性的誘因）假陽(yáng)性的去除 通過(guò)序列分析去除 質(zhì)粒消除實(shí)驗(yàn)剔除假陽(yáng)性 重新轉(zhuǎn)化含有誘餌質(zhì)粒的酵母株或不含誘餌質(zhì)粒的酵母株，分析兩種情況后去除假陽(yáng)性 利用一個(gè)不相關(guān)的蛋白作為誘餌蛋白測(cè)試相互作用 兩不或多步篩選酵母雙雜交的優(yōu)點(diǎn) 快速獲得相互作用蛋白的基因 只需單一步驟的質(zhì)粒構(gòu)建 在體內(nèi)鑒定蛋白的相互作用 弱小的，暫時(shí)的相互作用也能鑒定 能在整個(gè)時(shí)間段積聚弱小的相互作用信號(hào)酵母雙雜交應(yīng)用舉例 蛋白級(jí)聯(lián)底物的鑒定 鈣調(diào)蛋白與L-異天冬氨酰甲基轉(zhuǎn)移酶的相互作用 E2F1 突變子的遺傳特性 多肽荷爾蒙受體

5、相互作用 Pha-4在線(xiàn)蟲(chóng) C. elegans 中 的相互作用參考文獻(xiàn)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 t

6、o 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.

7、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: Clonin

8、g 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

9、, 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 continuedVidal, Marc, P. Braun, E. Chen, J

10、. 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 (19