Slide 11
Slide 12
Slide 13
GST-pull down原理
RIGI
Experimental
(GST-Fusion) GST
MAVS
Control
(GST) GST
GST
MAVS
RIGI
RIGI
GST
Wash SDS-PAGE
GST沉降示意图
Slide 14
Cross-linking
蛋白相互作用与酵母双杂交
Protein-protein Interaction & Yeast Two-hybrid
黄宝玉 2012.8.17
Slide 1
报告内容： 1.蛋白相互作用与研究方法 2.酵母双杂交原理和方法 3.酵母双杂交的发展 4.已有实验的进展 5.存在问题与下一步计划
Slide 2
Protein-protein Interaction
• 蛋白质之间相互作用以及通 过相互作用而形成的蛋白复 合物是细胞各种基本功能的 主要完成者。 • 几乎所有的重要生命活动， 包括DNA的复制与转录、蛋 白质的合成与分泌、信号转 导和代谢等等，都离不开蛋 白质之间的相互作用。
Slide 3
蛋白质相互作用研究技术 Genetic
Slide 30
测序正确的质粒转化酵母
Transformation of Competent Yeast Cells:
1. 加入质粒DNA 鲑鱼精DNA(变性的，10mg/ml) 加入感受态细胞, 轻弹混匀 加入PEG/LiAc, 轻弹混匀
2. 30℃水浴，每10轻弹混匀 3. 加入DMSO, 轻弹混匀 4. 42℃水浴，每5min轻弹混匀 5. 离心，弃上清，10000rpm 6. 用0.9% NaCl重悬
Slide 24
利用酵母双杂验证两蛋白相互作用实验流程
基因序列克隆
酵母质粒双酶切线性化
融合酶融合
转化大肠杆菌感受态并测序
测序正确的质粒转化酵母
涂板单缺培养基
挑选发育良好菌落mating
涂布二缺培养基
涂布四缺培养基
Slide 25
基因序列克隆
Amplify your bait insert by PCR using oligos that contain a 24bp homology to your bait, and a 15bp homology to the linear ends of pGBKT7, which are designed as follows: Forward Primer (111 = first codon of your bait)
Slide 20
三个启动子，四个报告基因
HIS3. When bait and prey proteins interact, Gal4-responsive His3 expression permits the cell to biosynthesize histidine and grow on –His minimal medium. ADE2.When two proteins interact, Ade2 expression is activated, allowing these cells to grow on –Ade minimal medium. AUR1-C. A dominant mutant version of the AUR1 gene that encodes the enzyme inositol phosphoryl ceramide synthase.its expression confers strong resistance (AbAr) to the otherwise highly toxic drug Aureobasidin A. MEL1. MEL-1 encodes α-galactosidase. As a result of two-hybrid interactions, α-galactosidase (MEL1) is expressed and secreted by the yeast cells. Yeast colonies that express Mel1 turn blue in the presence of the chromagenic substrate X-a-Gal.
Promoter
reporter gene
AD
Y
GAL4 UAS Promoter reporter gene
AD
X
DNA-BD
Y
Promoter
transcription
reporter gene
GAL4 UAS
(Fields&Song,1989)
Slide 19
酵母双杂交系统:三个启动子，四个报告基因
Slide 6
Solution phase selection with biotinylated antigen
antigen biotin
Slide 7
Bind to Streptavidin
coated microtitre wells
Slide 8
Wash to remove unbound phage particles.
Slide 31
涂布单缺培养基
pGADT7 Amp+ -Leu pGBKT7 Kan+ -Trp
Slide 32
挑选发育良好菌落mating并随后二缺四缺 培养基筛选
Slide 33
酵母双杂交系统的自激活验证
一般情况下，单独的 BD 可以与 GAL4 上 游活化序列（GAL UAS）结合，但不能引 起转录。然而，将一段具有转录激活活性 的转录因子基因构建到BD载体上，若其表 达产生的 BD 单独与 UAS 结合也可以引起 下游报告基因的转录，那么就称之为酵母 双杂中的自激活现象。
100-500ng 5ul 50ul 500ul
30min 20ul 15min 15s 1ml
7 取适量菌液涂布在相应的筛选培养基上。
我们采用的是PEG/ LiAc法转化酵母。 PEG是一种高分子聚合物, 在酵母 转化中起到在高浓度醋酸锂环境中 保护细胞膜,减少醋酸锂对细胞膜结 构的过度损伤,同时促进质粒与细胞 膜接触更紧密。 LiAc可使酵母细胞产生一种短暂的 感受性状态,此时它们能够摄取外源 性DNA 。DMSO增加细胞通透性以增 加转化效率。 鲑鱼精carrier DNA 为短的线形单 链DNA ,在转化实验中主要是保护质 粒免于被DNA 酶降解;另外还可能在 酵母细胞摄取外源性环形质粒DNA 中发挥协助作用。在每次使用前务 必进行热变性,使可能结合的双链 DNA 打开,保证鲑鱼精carrier DNA 在 转化实验体系中以单链形式存在。
5’-C ATG GAG GCC GAATTC 111 222 333 444 555 666 777 888
Reverse Primer (LLL = reverse complement of last codon of your bait) 5’-GC AGGTCGACGGATCC LLL NNN NNN NNN NNN NNN NNN NNN NOTE: These primers actually contain 16 bp of homology in order to keep the BamH I and EcoR I sites intact
Slide 34
原理：
GAL4BD Transcription factors
X
X
rr e e g p n o e t e r
Slide 35
酵母双杂交的毒性验证：
You should demonstrate that your bait protein is not toxic when expressed in yeast. If your bait is toxic to the yeast cells, both solid and liquid cultures will grow more slowly. If expression of your bait protein does have toxic effects, you may wish to switch to a vector (such as pGBT9) that has a lower level of expression. 1. Materials: • Y2HGold competent cell • SD/–Trp agar plates • SD/–Trp broth 2. Transform 100 ng of the following vectors: • pGBKT7 (empty) • pGBKT7 + cloned bait gene 3. Spread 100 μl of 1/10 and 1/100 dilutions of your transformation mixtures onto SD/–Trp. 4. Grow at 30°C for 3–5 days: Note : If your bait is toxic, you may notice that colonies containing your bait vector are significantly smaller than colonies containing the empty pGBKT7 vector.
Yeast Two-hybrid Phage Display Mutational analysis
Chemical
Crosslinking Label-transfer FeBABE mapping
Biochemical