Abstract: The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E.

Abstract: We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E.

Abstract: The present invention provides functional annotation of novel genes by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. The instant invention also provides an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among network component proteins are detected and probed with stimulators and inhibitors of the network and subcellular location of the interacting proteins is determined. Additionally, applicants' use this strategy to map a signal transduction network that controls the G0 to G1 transition in eukaryotes. Analysis of 148 combinations of 65 protein pairs in mammalian cells allows applicants' to propose a model of network architecture. The results demonstrate the feasibility of employing this strategy in genome-wide functional annotation efforts.

Abstract: The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: The present invention describes rapid methods to screen for biomolecular interactions in vivo based on protein fragment complementation assays (PCA). We have demonstrated an in vivo library-versus-library screening strategy that has numerous applications in the identification of novel protein-protein interactions and in directed evolution. Also we demonstrate the detection of protein-protein interactions starting with defined (full-length) cDNAs, and the concomitant generation of functional assays that provide initial validation of the cDNA products as being biologically relevant. Also, we screened a large cDNA collection using automated PCA, combined with quantitative detection of protein-protein complexes. The invention enables bait-vs.-library, library-vs.-library and defined gene screening in any type of cell or cellular context, and using a wide range of reporters and detection methods.

Abstract: We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E.

Abstract: The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: The present invention provides protein single-color and multi-color protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter such as monomeric enzymes and fluorescent proteins, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. The development of such assays provides for a broad, flexible and biologically relevant platform for drug discovery.

Abstract: The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule with the proviso that said protein is not ubiquitin.

Abstract: The present invention describes rapid and efficient methods to screen for biomolecular interactions in vivo based on protein fragment complementation assays (PCA). Examples are given that demonstrate the utility of the invention and the specific advantages of PCA that are not met by other library screening methods. In a first example, we demonstrate an in vivo library-versus-library screening strategy that has numerous applications in the identification of novel protein-protein interactions and in directed evolution. In another example we demonstrate the detection of protein-protein interactions starting with defined (full-length) cDNAs, and the concomitant generation of functional assays that provide initial validation of the cDNA products as being biologically relevant. In yet another example we demonstrate cDNA library screening in mammalian cells using a bait-vs.-library strategy combined with fluorescence detection.

Abstract: The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins.

Abstract: We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E.

Abstract: We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E.

Abstract: We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to-GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (lie to Val, Ala and Gly) resulted in a sequential increase in E.

Abstract: The present invention provides functional annotation of novel genes by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. The instant invention also provides an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among network component proteins are detected and probed with stimulators and inhibitors of the network and subcellular location of the interacting proteins is determined. Additionally, applicants' use this strategy to map a signal transduction network that controls the Go to G1 transition in eukaryotes. Analysis of 148 combinations of 65 protein pairs in mammalian cells allows applicants' to propose a model of network architecture. The results demonstrate the feasibility of employing this strategy in genome-wide functional annotation efforts.

Abstract: The invention provides a general protein-fragment complementation assays to detect biomolecular interactions in vivo and in vitro. The protein-complemetation assay/universal reporter system can be used to detect and screen an agonist and an antagonist of a membrane receptor system. The assay can be used to study protein-protein, protein-DNA, protein-RNA, protein-carbohydrate, and protein-small molecule interactions. The assay can be used to screen cDNA libraries for binding of a target protein with unknown proteins or libraries of small organic molecules for biological activity.