Improving yeast two-hybrid screening systems.

Yeast two-hybrid (Y2H) screening methods are an effective means for the detection of protein-protein interactions. Optimisation and automation has increased the throughput of the method to an extent that allows the systematic mapping of protein-protein interactions on a proteome-wide scale. Since two-hybrid screens fail to detect a great number of interactions, parallel high-throughput approaches are needed for proteome-wide interaction screens. In this review, we discuss and compare different approaches for adaptation of Y2H screening to high-throughput, the limits of the method and possible alternative approaches to complement the mapping of organism-wide protein-protein interactions.     

Analysing protein-protein interactions with the yeast two-hybrid system

Plant research is moving into the post-genomic era. Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives. All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights. One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system. This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo. In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology.     

酵母双杂交筛选与果蝇C(2)M相互作用的蛋白

同源染色体联会时形成的联会复合体(Synaptonemal complex, SC)是由减数分裂前期Ⅰ多种蛋白质聚集而成的超级复合结构。生殖细胞特异性的核蛋白C(2)M(Crossover suppressor on 2 of Manheim)在染色体上高度聚集可以诱导SC的形成。本文采用酵母双杂交方法,利用C(2)M的诱饵表达载体筛选果蝇cDNA文库,共发现40个可能与C(2)M相互作用的蛋白,包括多种DNA及组蛋白结合蛋白、ATPase、转录调节因子。从筛选的结果中,选取wech和Psf1基因构建了转基因果蝇,并在生殖细胞中进行了基因沉默,结果显示联会复合体的消失受到延迟。上述结果表明Wech和Psf1蛋白可能与C(2)M形成复合物,共同参与联会复合体的形成或其稳定性的维持。     

酵母双杂交系统及其应用

酵母双杂交系统（yeast two-hybrid system)是直接于细胞内研究蛋白质间相互作用的一种灵敏度很高,且非常有效的遗传学方法,在不同研究领域中广泛应用,并不断完善及改进,发展出单杂交系统（one-hybid system)和三杂交系统（three-hybrid system)等一系列相关的技术,克服了原系统的局限,本文对双杂交系统的原理,发展概况,应用,存在问题和前景等进行了综述。     

应用酵母双杂交技术寻找与GGNBP相互作用的蛋白质

生殖细胞缺陷症(gcd)小鼠突变体是20个世纪90年代初发现的一种不育突变小鼠,其不育原因是由于其胚胎期原始生殖细胞的数目低于正常。FancL(也叫Pog)的缺失是引起gcd突变小鼠的原因,FancL基因缺失后可能影响了小鼠胚胎期原始生殖细胞的增殖／存活和成年期小鼠精母细胞的减数分裂。FANCL是一种含有PHD结构域的泛素E3连接酶,是Fanconi贫血复合物的组分之一。在生殖细胞中,FANCL与GGN1和GGN3相互作用,GGN1和GGN2又与一种新的蛋白质GGNBP特异作用。但GGNBP蛋白的功能还不清楚。为了研究GGNBP的功能以及揭示更多的参与该过程的蛋白质,运用Clontech公司新开发的第3套酵母双杂交系统,以GGNBP为诱饵从成年小鼠睾丸cDNA库中筛选与其相互作用的蛋白质基因,发现了一个主要在睾丸中表达的新的基因,其编码的蛋白质产物在酵母系统中与GGNBP特异作用。     

利用酵母双杂交系统在人脑cDNA文库中筛选与禽流感病毒核蛋白相互作用的蛋白质

禽流感病毒核蛋白 (NP) 在病毒的转录、复制以及决定病毒的宿主特异性方面都具有重要作用。通过酵母双杂交系统筛选与核蛋白相互作用的蛋白,为进一步了解NP蛋白与细胞内蛋白质的相互关系以及流感病毒与宿主的相互关系奠定基础。应用酵母双杂交系统,构建NP诱饵质粒,进而筛选人脑cDNA文库,寻找可能与禽流感病毒NP相互作用的蛋白质。经过酵母双杂交共验证,得到7个与NP相互作用的阳性克隆。该结果为深入了解病毒复制的分子机理及其在蛋白质水平上与宿主蛋白的相互作用关系提供了线索。     

酵母双杂交筛选与GsCBRLK相互作用的蛋白质

GsCBRLK(calcium/calmodulin-binding receptor-like kinase from Glycine soja)在ABA及盐胁迫诱导的钙离子信号通路中起到关键的调节作用。为深入研究GsCBRLK蛋白的作用机制, 文章采用膜酵母双杂交系统, 以GsCBRLK为诱饵蛋白, 筛选与其相互作用的蛋白质。通过构建野生大豆盐胁迫条件下的cDNA文库、膜酵母双杂交系统筛选、复筛、回转验证、生物信息学分析以及酵母体内互作验证等手段, 最终获得2个(SNARE 和 14-3-3 蛋白)与GsCBRLK诱饵蛋白相互作用的蛋白质。     

发展中的酵母杂交体系

酵母杂交体系包括双杂交、反向双杂交和三杂交等体系。双杂交作为一种新兴的体内研究蛋白质之间相互作用的方法,已经得到了广泛的应用。而反向双杂交和三杂交系统是在双杂交基础上发展起来的两种新技术。反向双杂交除了筛选突变株,以获取蛋白质结合的信息外,还能发现可导致已知蛋白质间特异相互作用发生解离的肽类或其他小分子物质,进一步分析蛋白质间作用位点、调控。三杂交系统则在蛋白质与小分子配基之间以及多种蛋白质之间相     

Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.     

神经元蛋白3.1结合蛋白酵母双杂交筛选体系的建立

神经元蛋白3.1(P311)是肺泡发育的上游调节因子。以pEGFP-P311重组质粒为模板,利用PCR方法扩增P311基因编码序列。通过Nde I和BamH I位点插入诱饵载体pGBKT7,构建重组诱饵载体pGBKT7-P311。重组体转化酵母菌AH109进行自激活和毒性检测,结果 DNA-BD-P311融合蛋白无单独激活报告基因作用,对酵母菌亦无毒性。以出生11 d小鼠肺组织为材料,提取总RNA。逆转录产生单链cDNA,通过长距离PCR进行扩增。扩增产物ds cDNA电泳后可见大小为0.2～3.0 kb间的弥散状分布条带,说明文库cDNA可满足筛选要求。诱饵载体pGBKT7-P311的构建及相应小鼠肺组织cDNA文库的建立,为进一步利用酵母双杂交技术探讨P311功能奠定了基础。     

用酵母双杂交系统研究SMAD3和SMAD4的相互作用

利用酵母双杂交试验,鉴定了细胞内信号传导蛋白SMAD3和SMAD4的相互作用。通过SMAD3和SMAD4各突变体的同源和异源相互作用的双杂交反应,确定SMAD4介导信号传递的功能区在中间连接区,SMAD3的功能区在C末端。     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

用酵母双杂交系统研究胰岛素样生长因子-1突变体与其结合蛋白-3的相互作用

为研究胰岛素样生长因子 1(IGF1)及其突变体与IGF结合蛋白 3(IGFBP3)的相互作用 ,针对IGF1的第 3、4、15、16位氨基酸残基 ,采用定点突变的方法构建了 [Y15L16 ]IGF1和 [Q3A4Y15L16 ]IGF1。然后分别将IGF1/IGF1突变体和IGFBP3cDNA克隆至酵母表达载体pGBT9和pACT2中 ,利用酵母双杂交技术检测IGF1/IGF1突变体和IGFBP3之间的相互作用。结果表明用酵母双杂交系统检测IGF1与其结合蛋白的结合力是可行的 ,构建的这两个IGF1突变体与IGFBP3的结合力 ,与天然IGF1相比 ,结合力大大减小     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast     

中华蜜蜂幼虫膜蛋白酵母双杂交文库的构建

【目的】本研究旨在利用位点特异性重组技术(FullCoV)将中华蜜蜂 Apis cerana cerana 幼虫膜蛋白cDNA连接到pPR3-N载体上,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库。【方法】提取2-3日龄中华蜜蜂工蜂幼虫总RNA;分离mRNA后,在反转录酶的作用下合成幼虫膜蛋白cDNA第1链,并合成双链cDNA。在双链cDNA的5′端加上带有重组序列的接头后,通过FullCoV技术与载体pPR3-N进行连接,然后将连接产物电转化到DH10B感受态细胞,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,并对该文库插入片段大小和文库滴度进行检测。【结果】通过FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,经检测,中华蜜蜂幼虫膜蛋白酵母cDNA文库的总库容量为1.5×10^7 cfu,文库滴度为3×10^6 cfu/mL,重组率达到100%。【结论】本研究利用FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母cDNA文库,为进一步探究感染中华蜜蜂的病原微生物与宿主蛋白互作研究奠定了基础。     

Screening of proteins that interact with human thrombopoietin receptor c-Mpl using yeast two-hybrid system

Thrombopoietin (TPO) is the major cytokine involved in platelet production and exerts its effects via the receptor c-Mpl. The yeast two-hybrid system has been used to screen the proteins interacting with c-Mpl. First, the cDNA fragment of c-Mpl intracellular domain was cloned into two-hybrid vector pAS2, and the resulting plasmid is designated as pASMM. Then a human placenta cDNA library was screened using the pASMM as a target plasmid. Seven positive clones were isolated from 150 000 independent transformants. Sequence analysis of one of the positive clones demonstrates that a part of coding sequence of vimentin from 611 bp to 3′ end and flanking non-translation region was obtained. Therefore, there is an interaction between vimentin and TPO receptor. The results suggest that cytoskeletal protein may play an important role in TPO signal transduction pathway.