大肠杆菌杂交系统及应用 演讲人：冯燕丽 2003年4月25日.

Presentation on theme: "大肠杆菌杂交系统及应用 演讲人：冯燕丽 2003年4月25日."— Presentation transcript:

1 大肠杆菌杂交系统及应用 演讲人：冯燕丽 2003年4月25日

2 原理 依赖转录激活过程； 报告基因的表达水平与bait（已知蛋白）与 target （已知蛋白）蛋白间作用强度呈正相关性；
双杂交系统：筛选已知相互作用的蛋白 或与特定蛋白作用的未知蛋白 生化方法：需要纯化蛋白或抗体

3 方法 材料：bait、target载体 报告细胞 其他必要因素 要求：bait载体，pBT , 3.1kb,编码全 长细菌噬菌体cl蛋白
target载体,pTRG, 4.3kb,控制 RNA 多聚酶 亚基和连接区的转录 报告细胞,XL1-Blue MRF’,无任何限制系统,与cDNA文库建立方法兼容

4 已知蛋白(bait)  全长细菌噬菌体蛋白cI cI（N-端DNA结合区和C-端二硫基）
结合位置：在操纵子序列的上游 目标蛋白（target）RNA 多聚酶 亚基N-端 报告细胞中进行: bait  target 作用后启动两个报告基因ampr、lacZ 具体: bait与 target作用时，其恢复和稳定RNA 多聚酶 启动子的结合，激活报告基因（AmpR）转录 ； 报告基因 半乳糖苷酶基因，同一启动子，通过 可见的表型变化来证实bait与 target间的相互作用

5 另一系统 依赖信号转导途径的重建； 利用两蛋白间的作用导致片断间的功能性互补，从而引起cAMP 合成。cAMP再激活分解代谢操纵子（乳糖或麦芽糖）转录，产生可见的特征性的表型变化。

6 Fig. 1.   Principle of an E. coli two-hybrid system based on functional complementation of CyaA fragments. (Upper) Schematic of the basic principle of in vivo complementation between the two fragments of the catalytic domain of B. pertussis adenylate cyclase. The two boxes represent the T25 and T18 fragments corresponding to amino acids and of the CyaA protein. In A, the full-length catalytic domain (residues 1-399), when expressed in E. coli, exhibits a basal calmodulin-independent activity that results in cAMP synthesis. In B, the two fragments, T25 and T18, when coexpressed as independent polypeptides, are unable to interact and no cAMP synthesis occurs. In C, the two fragments, fused to two interacting proteins, X and Y, are brought into close proximity, resulting in functional complementation followed by cAMP production. (Lower) Schematic of the readout of the complementation. cAMP, synthesized in an E. coli cya strain by the complementing T25 and T18 pairs, binds to the catabolite gene activator protein, CAP. The cAMP/CAP complex then can recognize specific promoters and switch on the transcription of the corresponding genes. These reporter genes can be either natural E. coli genes, such as lacZ or mal genes, or synthetic ones, such as antibiotic-resistance genes fused to a cAMP/CAP-dependent promoter.

7 材料：DHP1 （E.coli cya菌株，腺苷酸环化酶缺失型） 表达载体pCm-AHL1
pT25p、pT18(含两个互补片断T25、T18，组成 Bordetella pertussis腺苷酸环化酶的媒触区）

8 方法： 1、设计功能互补的CyaA的T25和T18片断； 2、用功能互补反应在体内检测蛋白间作 用 pT25p、pT18共转DHP1；
在加入maltose 中培养； 检测：全表达呈红色，不表达呈白色 2、用功能互补反应在体内检测蛋白间作 用

9 Fig. 2. Schematic representation of plasmids
Fig. 2.   Schematic representation of plasmids. The open boxes represent the ORFs of -lactamase (bla) and chloramphenicol acetyl transferase (cat) genes. The solid boxes correspond to the ORF of cyaA', with codon numbers indicated below. The hatched boxes correspond to the multicloning site sequences (MCS) that are fused at the indicated position of the cya ORF. The origin of replication of the plasmids is indicated by shaded boxes.

10 表1 Analysis of complementation in DHP1 strain
Plasmids Phenotype on MacConkey/maltose None white pCm-AHL red/24hr pT25pT white/72hr pT25pT18-zip white/72hr pT25-zippT white/72hr pT25-zippT18-zip red/24hr

11 表2 Complementation between various chimeric protein
Plasmids Phenotype on MacConkey/maltose pT25-TyrpT18 -Tyr red/40hr pT25 -Tyr pT white/96hr pT25pT18 –Tyr white/96hr pT25-Tyr pT white/96hr pT25-zippT18 –Tyr white/96hr pT25-prp11pT18 –prp red/40hr 说明：互补受整合到T25、T18的多肽的特异性识别控制 嵌合蛋白的互补作用也依赖其配体的特定性作用

12 Fig. 3.   Screening of interacting proteins with the bacterial two-hybrid system. DHP1 cells were cotransformed with a mixture of plasmids pTI8, pT18-zip, and pT18-Tyr and either pT25 (A) or pT25-zip (B), plated on LB-X-gal agar plates containing 0.5 mM isopropyl- -D-thiogalactopyranoside, ampicillin, and chloramphenicol, and incubated for 30 hr at 30°C. Note that the cya+ colonies are larger than the cya ones.

13 结论 优势：与酵母双杂交系统的比较 * 决定细菌杂交系统能更灵敏检测难发现特定蛋白结合体 对待测蛋白的特性要求低，因此可适合更大蛋白库筛选
传统酵母双杂交系统 BacterioMatch双杂交系统 生长时间 天 天或过夜培养 转化率 DNA操作过程 额外转化过程到E.coli 直接克隆筛选 DNA分离 酵母染色体DNA与质粒共沉淀 简单、方便的制备 （去除了一步转化） 克隆分析 繁琐的纯化后进行 直接进行 结果评价 假阳性多 假阳性少 * 决定细菌杂交系统能更灵敏检测难发现特定蛋白结合体 对待测蛋白的特性要求低，因此可适合更大蛋白库筛选

14 应用 用于研究蛋白-DNA、蛋白-蛋白间相互作用

15 Fig. 1. (A) Transcriptional activation in a previously described E
Fig. 1.   (A) Transcriptional activation in a previously described E. coli-based genetic screen [developed by Hochschild and coworkers (8, 10)] for studying protein-DNA and protein-protein interactions. (B) Modified reporter template for our E. coli-based genetic selection system. (C) Model for transcriptional activation of the Pzif promoter by fusion proteins Gal11P-Zif123 and Gal4. ZF1, ZF2, and ZF3 are the three zinc fingers of the Zif268 protein. (Although Gal11P-Zif123 efficiently activates our Pzif promoter, we note that the spacing between the Zif268-binding site and the transcription start site has not yet been optimized.)

16 Fig. 2.   An E. coli-based selection system for identifying zinc finger variants from large randomized libraries. (Left) A selection strain cell bearing a randomized zinc finger (white oval) that is unable to bind the target DNA subsite of interest (black box). This candidate fails to activate transcription of the weak promoter controlling HIS3 expression and therefore cells expressing this candidate fail to grow on HIS-selective medium. (Right) A library candidate bearing a particular zinc finger (one member of the randomized library) (black oval) that can bind the target DNA site. This candidate can activate HIS3 expression and therefore cells expressing this candidate grow on HIS-selective medium.

17 Fig. 3.   Recognition helix sequences of fingers isolated by our selection. For candidates that were isolated multiple times (as judged by nucleotide sequence), the number of clones obtained is shown in parentheses. The consensus sequence(s) of fingers selected by phage display for each target subsite also are shown (6). +, positively charged residue; _, no discernible preference; *, candidates with a 2-bp deletion downstream of the sequence encoding the recognition helix; and arrows illustrate a few of the most plausible potential base contacts.

18 Thank a lot!