A two-hybrid dual bait system to discriminate specificity of protein interactions.

Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.     

细菌双杂交系统的改进

解析蛋白质之间的相互作用,对理解调控和代谢等生物学过程具有重要意义。其中基于互补腺苷酸环化酶功能的细菌双杂交系统是一种研究蛋白质之间相互作用的有效手段。本文在利用该系统时发现存在假阳性高的缺陷。进一步报告基因活性分析表明产生假阳性的原因是不同克隆的生理状态影响了LacZ的输出,即lacZ启动子除了受cAMP信号分子的控制,还受到其他调控蛋白的直接或间接的调控。为了降低生理因素对报告基因的影响,我们向该系统引入用多拷贝lacZ启动子控制的gfp报告基因。这不仅通过增加lacZ启动子的数量,弱化了生理因素的影响;还实现了第二个报告基因gfp与lacZ同时报告蛋白质之间的相互作用情况,提高了输出的准确性。系统的实验验证表明,我们改进的细菌双杂交系统的灵敏性确实得到大幅提高。     

Streamlined yeast colorimetric reporter activity assays using scanners and plate readers

Two-hybrid systems have become favored tools for detection and analysis of protein interactions because of their low cost and ease of use compared to biochemical or biophysical interaction technologies. It is possible to augment the utility of two-hybrid systems and derivative systems such as dual-bait two-hybrid systems by adapting strategies that speed the analysis of the relative strength of a series of protein-protein associations. This report describes two simple techniques that employ either a flatbed scanner or a plate reader to quantitate the activity of colorimetric reporters such as LacZ or GusA commonly used in two-hybrid approaches.     

Illuminating the Dynamics of Intracellular Activity with 'Active' Molecular Reporters

Traditionally, fluorescent and luminescent reporter proteins have been used as indicators of gene expression and protein localization. However, insightful mutagenesis and protein engineering strategies have transformed these simple passive reporters into active biological sensors. Molecular reporters are now being designed to alter their intrinsic optical properties in response to specific biomolecular interactions. Applications for these novel biological sensors range from monitoring intracellular pH and ion fluxes to detecting protein-protein interactions and enzymatic activity. The ability to monitor the dynamics of intracellular activity in response to external stimuli can help elucidate the cascade of events involved in complex processes such as mechanotransduction. Here we review some of the approaches used to create these novel biological sensors, including resonance energy transfer (RET) between reporter proteins and protein fragmentation strategies.     

A novel triple fusion reporter system for use in gene trap mutagenesis

Gene trapping is an insertional mutagenesis strategy that allows for simultaneous gene identification and mutation in embryonic stem (ES) cells. Gene trap vectors both disrupt coding sequence and report on the genes' endogenous expression. The most popular gene trap reporter to date combines beta-galactosidase expression with neomycin resistance in a fusion protein known as beta-geo. Here we describe a refinement to this reporter that also incorporates real time fluorescent readouts. We have constructed a series of gene trap vectors incorporating a novel tripartite fusion protein consisting of EGFP, beta-galactosidase, and the neomycin or hygromycin resistance activities. Our results indicate that these triple fusions can function efficiently as reporters of endogenous trapped gene expression and subcellular localization. We show that these fusion proteins constitute versatile gene trap reporters whose activity can be detected in real time by fluorescence and in fixed tissue with a sensitive enzymatic activity.     

A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

Despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. Protein complementation assays (PCAs) are commonly used to study interactions between proteins due to their simplicity. They are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. Recently, a protein complementation method named split-TEV (tobacco etch virus) has been developed and is based on the functional reconstitution of TEV protease and subsequent proteolytic-mediated activation of reporters. In this work, we have developed a modification of the split-TEV method to study the interactions between membrane proteins with increased specificity. This assay was validated by addressing the interactions between different membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels. By comparing it with another PCA, we found that this new method showed a higher sensitivity.     

Counter-selectable marker for bacterial-based interaction trap systems

Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F' episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart.     

A single-chain antibody/epitope system for functional analysis of protein-protein interactions

Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.     

Approaches to detecting false positives in yeast two-hybrid systems

While many novel associations predicted by two-hybrid library screens reflect actual biological associations of two proteins in vivo, at times the functional co-relevance of two proteins scored as interacting in the two-hybrid system is unlikely. The reason for this positive score remains obscure, which leads to designating such clones as false positives. After investigating the effect of over-expressing a series of putative false positives in yeast, we determined that expression of some of these clones induces an array of biological effects in yeast, including altered growth rate and cell permeability, that bias perceived activity of LacZ reporters. Based on these observations, we identify four simple strategies that can assist in determining whether a protein is likely to have been selected in a two-hybrid screen because of indirect metabolic effects.     

Identification of binary interactions between human cytomegalovirus virion proteins

Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis.     

细菌双杂交系统的优化及其应用

细菌双杂交系统是一种用于检测体内蛋白质互作的方法,该方法互补腺苷酸环化酶功能,通过检测细胞表达的β-半乳糖苷酶LacZ的活性,分析蛋白质互作能力.但在应用过程中,发现存在操作繁琐、灵敏度低、难实现高通量操作等缺陷.本研究目的是对原有细菌双杂交进行优化,建立一种操作方便、可批量操作、具有较高灵敏度和能够实现实时监测的细菌...