蛋白相互作用酵母双杂交

antigen biotin
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Bind to Streptavidin
coated microtitre wells
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Wash to remove unbound phage particles.
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Elute bound phage
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Amplify eluted phage
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。
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蛋白质相互作用研究技术
Repeat selection
Analyze a) ELISA b) Specificity c) Sequencing d) Affinity e) Activity
感染 和扩增
E.coli
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GST-pull down原理
RIGI
Experimental Control
30min
3. 加入DMSO, 轻弹混匀
20ul
4. 42℃水浴，每5min轻弹混匀
15min
5. 离心，弃上清，10000rpm
15s
6. 用0.9% NaCl重悬
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。
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酵母质粒双酶切线性化
10×NEB buffer 100×BSA EcoR I BamH I pGBKT7
1ul 0.1ul 0.2ul 0.2ul 8.5ul
37℃，3h
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融合酶融合
5×In-Fusion HD Enzyme Premix Linearized Vector(50-200ng) Purified PCR Fragment(10-200ng) dH2O
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原理：
GAL4BD
Transcription factors
X
X r e p o r t e r g e n e
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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.
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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
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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) 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
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。
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酵母双杂交系统的功能
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酵母双杂交的简单介绍
酵母双杂交原理图
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酵母双杂交系统的原理
X
DNA-BD
GAL4 UAS
Promoter
GAL4 UAS
Promoter
reporter gene
AD
Y
reporter gene
X
DNA-BD
GAL4 UAS
AD
Y
Promoter
transcription
reporter gene
(Fields&Song,1989)
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酵母双杂交系统:三个启动子，四个报告基因
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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, αgalactosidase (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.
Genetic
Yeast Two-hybrid Phage Display Mutational analysis
Chemical
Crosslinking Label-transfer FeBABE mapping
Biochemical
Immunoprecipitation (IP) Co-Immunoprecipitation
(Co-IP) Pull-Down Assays Far Western
Fluorescent
Immunofluorescence co-localization
FRET (Fluorescence Resonance Energy Transfer)
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噬菌体展示技术的原理
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酵母双杂交所用的两个质粒载体
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Restriction Map and Multiple Cloning Site (MCS) of pGBKT7