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Stanley Fields - One of the best experts on this subject based on the ideXlab platform.
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the yeast Two Hybrid System
1997Co-Authors: Paul L Bartel, Stanley FieldsAbstract:The yeast Two-Hybrid System is among the most powerful and generally useful methods for screening genes that are functional in various physiological processes and Systems, and for determining gene interactions. Fields is one of the pioneers and leading practitioners of this approach, and the volume provides an accessible, critical summary of the methods and range of applications of gene screening possible with the yeast Two-Hybrid System.
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Protein-peptide interactions analyzed with the yeast Two-Hybrid System
Nucleic acids research, 1995Co-Authors: Meijia Yang, Stanley FieldsAbstract:The yeast Two-Hybrid System was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, all of which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay varied over a 40-fold range. Mutagenesis of the DNA encoding Two of these peptides followed by screening in the Two-Hybrid System allowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants to Rb, determined by surface plasmon resonance, correlated with results from the Two-Hybrid assay. This method offers several advantageous features compared to existing technology for screening peptide libraries: in vivo detection of protein-peptide interactions, high sensitivity, the capacity for rapid genetic screening to identify stronger and weaker binding peptide variants, and the use of a simple assay (transcriptional activity) as a means to assess binding affinity.
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[16] Analyzing protein-protein interactions using Two-Hybrid System
Methods in enzymology, 1995Co-Authors: Paul L Bartel, Stanley FieldsAbstract:Publisher Summary This chapter presents the analysis of protein–protein interactions using Two-Hybrid System. Protein-protein interactions play a critical role in most biological processes. The studies defining domains of proteins that are responsible for specific interactions have contributed significantly to unraveling the mechanisms of tumorigenesis. The Two-Hybrid System is a yeast-based genetic assay for detecting protein– protein interactions in vivo. It can be used to establish interactions between Two known proteins or to search genomic or cDNA libraries for proteins that interact with a target protein. For this latter application, the gene encoding the protein that interacts with a target protein is immediately available on a plasmid, which is not the case for many biochemical methods to detect interacting proteins. The Two-Hybrid System has also been used to define the protein domains that mediate an interaction and to identify specific residues that are involved in a protein–protein interaction. The chapter also discusses the basis for this method and presents the protocols that are necessary to use this System.
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the Two Hybrid System an assay for protein protein interactions
Trends in Genetics, 1994Co-Authors: Stanley Fields, Rolf SternglanzAbstract:The Two-Hybrid System is a yeast-based genetic assay for detecting protein-protein interactions. It can be used to identify proteins that bind to a protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA-binding proteins, to identify peptides that bind to a protein and, potentially, to screen for drugs.
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Elimination of false positives that arise in using the Two-Hybrid System.
BioTechniques, 1993Co-Authors: P. Bartel, Rolf Sternglanz, Cheng-ting Chien, Stanley FieldsAbstract:The Two-Hybrid System is a genetic method to identify proteins that interact with a specific target protein, which is expressed in yeast as a Hybrid with a DNA-binding domain. Use of this method entails transforming yeast, both with this DNA-binding domain Hybrid and with a library of activation domain Hybrids, followed by screening for transcriptional activation of a reporter gene. In addition to proteins that interact with the target protein, a number of false positives are identified. We provide possible explanations for these false positives along with rapid assays to eliminate them.
Daniel Auerbach - One of the best experts on this subject based on the ideXlab platform.
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The yeast Two-Hybrid System and its role in drug discovery.
Expert opinion on therapeutic targets, 2008Co-Authors: Nicolas Lentze, Daniel AuerbachAbstract:Background: The yeast Two-Hybrid System is the most widely used genetic assay to identify and characterize novel protein interactions. Over the past decade, the System has been adapted to cover an increasingly wide range of applications, including various tasks within the drug discovery and development process. Objective: We highlight the role of different Two-Hybrid Systems within the drug discovery process, including target identification and validation and the selection of affinity reagents for protein targets, such as peptides and small molecules. Methods: We have focused on applications where the Two-Hybrid System has been used to great advantage and have sought to put a special emphasis on less conventional but promising approaches, such as the identification of agents which block therapeutically relevant protein interactions. Conclusions: The yeast Two-Hybrid System has evolved from a method mainly used in basic research to a collection of versatile screening Systems with the potential to affect ma...
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Membrane-based yeast Two-Hybrid System to detect protein interactions.
Current protocols in protein science, 2008Co-Authors: Nicolas Lentze, Daniel AuerbachAbstract:The classical yeast Two-Hybrid System and its modifications have been successfully used over the past decade to investigate interactions between most classes of proteins expressed in a given cell or tissue. However, some proteins (e.g., integral membrane proteins or nuclear proteins) are relatively difficult to investigate by standard yeast Two-Hybrid methods either because they are retained at cellular membranes or they activate the System in the absence of a true protein interaction. The membrane-based yeast Two-Hybrid (MbY2H) System presented in this unit overcomes some of these limitations. It is based on the split-ubiquitin protein complementation assay and detects protein interactions directly at the membrane, thereby allowing the use of full-length integral membrane proteins and membrane-associated proteins as baits to hunt for novel interaction partners. A simple modification also allows the use of proteins that are self-activating in a classical yeast Two-Hybrid System (e.g., acidic proteins and many transcription factors). Like the yeast Two-Hybrid System, the MbY2H System can also be used for interaction discovery by screening complex cDNA libraries for novel interaction partners.
Daniel Ladant - One of the best experts on this subject based on the ideXlab platform.
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protein protein interaction between bacillus stearothermophilus tyrosyl trna synthetase subdomains revealed by a bacterial Two Hybrid System
Journal of Molecular Microbiology and Biotechnology, 2001Co-Authors: Gouzel Karimova, Agnes Ullmann, Daniel LadantAbstract:We have recently developed a bacterial Two-Hybrid System (BACTH), based on functional complementation between Two fragments of the catalytic domain of Bordetella pertussis adenylate cyclase (AC), that allows an easy in vivo screening and selection of functional interactions between Two proteins in Escherichia coli. In this work, we have further explored the potentialities of the BACTH System to study protein-protein interactions, using as a model, the interactions between various subdomains of the dimeric tyrosyl-tRNA synthetase (TyrRS) of Bacillus stearothermophilus. Using the BACTH System we confirmed the known interactions of the alpha/beta domains and those between the alpha/beta domain and the alpha domain that could be anticipated from the three-dimensional structure of TyrRS. Interestingly, the BACTH System revealed the unexpected interaction between the TyrRS alpha domains which is presumably mediated by a pseudo-leucine zipper motif. This study illustrates the interest of the bacterial Two-Hybrid System to delineate interacting domains of proteins and shows that it can reveal interactions that occur in vivo and that were not anticipated from the three-dimensional structure of the protein of interest.
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A bacterial Two-Hybrid System that exploits a cAMP signaling cascade in Escherichia coli.
Methods in enzymology, 2000Co-Authors: Gouzel Karimova, Agnes Ullmann, Daniel LadantAbstract:Publisher Summary Most biological processes involve specific protein–protein interactions. The yeast Two-Hybrid System represents a powerful in vivo approach to analyze interactions among macromolecules and screen for polypeptides that bind to a given bait protein. Bacterial equivalents to the yeast Two-Hybrid System have not been developed yet. This chapter describes a novel bacterial Two-Hybrid System that allows an easy in vivo screening and selection of functional interactions between Two proteins. This System, because of its sensitivity and simplicity, could have broad application in the studies of structure–function relationships in biological macromolecules, in the functional analysis of genomes, and in high-throughput screening of interacting ligands or new therapeutic agents.
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Bordetella pertussis adenylate cyclase toxin as a tool to analyze molecular interactions in a bacterial Two-Hybrid System.
International journal of medical microbiology : IJMM, 2000Co-Authors: Gouzel Karimova, Agnes Ullmann, Daniel LadantAbstract:Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin (CyaA) that is able to enter into eukaryotic cells. We took advantage of the modular structure of the catalytic domain of CyaA to design a genetic System that can detect protein-protein interactions in Escherichia coli. This bacterial Two-Hybrid System is based on the functional complementation between Two complementary fragments, T25 and T18, of the catalytic domain of CyaA, in an E. coli cya strain. This bacterial Two-Hybrid System could find applications in the studies of structure/function relationships of proteins, in functional analysis of genomes, in high-throughput screening of interacting ligands and in design of new therapeutic agents.
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a bacterial Two Hybrid System based on a reconstituted signal transduction pathway
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Gouzel Karimova, Josette Pidoux, Agnes Ullmann, Daniel LadantAbstract:We describe a bacterial Two-Hybrid System that allows an easy in vivo screening and selection of functional interactions between Two proteins. This genetic test is based on the reconstitution, in an Escherichia coli cya strain, of a signal transduction pathway that takes advantage of the positive control exerted by cAMP. Two putative interacting proteins are genetically fused to Two complementary fragments, T25 and T18, that constitute the catalytic domain of Bordetella pertussis adenylate cyclase. Association of the Two-Hybrid proteins results in functional complementation between T25 and T18 fragments and leads to cAMP synthesis. Cyclic AMP then triggers transcriptional activation of catabolic operons, such as lactose or maltose, that yield a characteristic phenotype. In this genetic test, the involvement of a signaling cascade offers the unique property that association between the Hybrid proteins can be spatially separated from the transcriptional activation readout. This permits a versatile design of screening procedures either for ligands that bind to a given “bait,” as in the classical yeast Two-Hybrid System, or for molecules or mutations that block a given interaction between Two proteins of interest.
Stephen J Elledge - One of the best experts on this subject based on the ideXlab platform.
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gene identification using the yeast Two Hybrid System
Methods in Enzymology, 1996Co-Authors: Chang Bai, Stephen J ElledgeAbstract:Publisher Summary This chapter discusses the gene identification using the yeast Two-Hybrid System. The yeast Two-Hybrid System was devised to identify genes encoding proteins that physically associate with a given protein in vivo. This is a versatile and powerful method that is applicable to most, if not all, proteins once their genes have been isolated. In contrast to biochemical methods detecting protein-protein interaction, this System is based on a yeast genetic assay in which the interaction of Two proteins is measured by the reconstitution of a functional transcription activator in yeast. Protein-protein interactions have attracted much attention because they form the basis of a wide variety of biochemical reactions. The identification of proteins that interact with a known protein is an essential aspect of the elucidation of the regulation and function of that protein. This interest has stimulated the development of a number of biochemical and genetic approaches to identify and clone genes encoding interacting proteins, including coimmunoprecipitation, copurification, cross-linking, and direct expression library screening using proteins as probes.
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finding prospective partners in the library the Two Hybrid System and phage display find a match
Trends in Biochemical Sciences, 1995Co-Authors: James B Allen, Stephen J Elledge, Mark W Walberg, Michael C EdwardsAbstract:Abstract The Two-Hybrid System uses the efficacy of yeast genetic assays to identify protein-protein interactions. It permits the rapid cloning of genes encoding products that interact with a given protein of interest. Also being developed are phage display methods that allow direct physical selection of binding proteins. These methods have significantly altered strategies for analysing signaling and regulatory pathways.
Li Zhu - One of the best experts on this subject based on the ideXlab platform.
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Mammalian Two-Hybrid System: A Complementary Approach to the Yeast Two-Hybrid System
BioTechniques, 1997Co-Authors: Ying Luo, Anna Batalao, Helen Zhou, Li ZhuAbstract:Here we demonstrate the use of a mammalian Two-Hybrid System to study protein-protein interactions. Like the yeast Two-Hybrid System, this is a genetic, in vivo assay based on the reconstitution of...
