Abstract: A method of screening biologically active agent based on the analysis of complex biological responses in culture. Methods for selecting cells and culture conditions for such screens are provided, as well as the identification of an optimized set of discrete parameters to be measured, and the use of biomap analysis for rapid identification and characterization of drug candidates, genetic sequences acting pathways, and the like. A feature of the invention is simultaneous screening of a large number of cellular pathways, and the rapid identification of compounds that cause cellular responses.

Abstract: Methods and materials are disclosed for use in an enzyme fragment complementation assay using complementary fragments of ?-galactosidase to study the trafficking of proteins in a cell. Compounds that bind to a target peptide have been found to affect protein folding and therefore trafficking. ?-Galactosidase fragments, an enzyme donor (ED) and an enzyme acceptor (EA), are fused to a target peptide and to an intracellular compartment protein, wherein the compartment is involved in intracellular trafficking. Contacting the cell with a compound that binds to the target peptide results in enhanced movement of the protein through the cellular trafficking pathway comprised of the endoplasmic reticulum, Golgi apparatus, the plasma membrane, endosomes, etc. Using this approach, compounds that bind to a target peptide and alter its ability to traffic through the normal cellular pathway can be readily detected.

Abstract: A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of ?-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of ?-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the ?-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.

Abstract: Methods and compositions are provided for assaying for enzymes capable of releasing an enzyme donor fragment (ED) conjugated to a nucleic acid strand bonded to a surface. Conveniently, the beads are magnetic allowing segregation of the beads during the determination. Upon addition of enzyme acceptor fragment (EA) and substrate to the assay mixture, the method allows for discrimination between ED free in solution and ED bound to the bead. The complexing of ED and EA provides an active ?-galactosidase enzyme. The method permits the assay of any substance involved in a pathway that can result in a reaction releasing the ED.

Abstract: Provided herein are assays useful, for example, for determining the activity of a protein involved in a cellular process. In some embodiments, the activity of the protein is assessed using a nucleic acid tag, and in particular, by detecting the presence of a nucleic acid tag. Such assays can be used, for example, to study the effects of test compounds as modulators, e.g., inhibitors, agonists and antagonists, of protein activity.

Abstract: Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation and the modulation of activation by a candidate compound are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of ?-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of ?-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a ?-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

Abstract: Truncated fragments of the small fragment of ?-galactosidase are provided that have low affinity for the large fragment of ?-galactosidase and provide for robust signals when two fusion proteins are complexed due to the binding of the proteins to which the ?-galactosidase fragments are fused. The truncated fragments do not interfere with the complexing of the two proteins and allow for the two proteins to function and be responsive to candidate compounds that affect complex formation.

Abstract: Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of ?-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of ?-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a ?-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

Abstract: A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of ?-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of ?-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the ?-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.

Abstract: Methods and genetic constructs are provided for detecting the binding of nuclear hormone receptors to a coactivator/corepressor. The methods employ enzyme fragment complementation using fragments of ?-galactosidase as the detection system. Cells are transformed to express the large fragment of ?-galactosidase fused to a member of the complex with NHR for initiation of transcription and have it localized in the nucleus and to express the small fragment of ?-galactosidase fused to the nuclear hormone receptor for binding to the member upon stimulation with a ligand.

Abstract: Sensitive assays for candidate compounds affecting GPCR activity are provided using a cell containing fusion proteins comprising a first fusion protein comprising (a) a target GPCR fused to a small fragment of ?-galactosidase through a linker comprising a phosphorylation site or (b) a GPCR or a protein of interest, where the GPCR and protein of interest form a complex and one of them is fused to the small fragment of ?-galactosidase; and a second fusion protein comprising arrestin fused to a large fragment of ?-galactosidase. In (a), the affinity of the small and large fragments is optimized based on the background to signal ratio and the absolute signal observed. The assay is performed using a ?-galactosidase substrate that provides a detectable optical signal.

Abstract: Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of ?-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of ?-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a ?-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

Abstract: Improved methods of determining the intracellular state of a protein as well as modifications of the protein are provided by introducing a surrogate fusion protein comprising a member of an enzyme fragment complementation complex and a target protein. After exposing cells transformed with the surrogate fusion protein to a change in environment, e.g. a candidate drug, the cells are lysed, the lysate separated into fractions or bands, conveniently by gel electrophoresis and transferring the proteins by Western blot to a membrane. The bands on the membrane are developed using the other member of the enzyme fragment complementation complex and a substrate providing a detectable signal. The method is found to provide high sensitivity and the ability to observe modifications of the target protein.

Abstract: An amplifiable ?-lactamase substrate is provided comprising an enzyme donor fragment of ?-galactosidase linked to form a ring to a ?-lactam ring that is a substrate for lactamase and upon opening of the ?-lactam ring the enzyme donor fragment becomes linearized. The cyclic substrate only weakly binds to the enzyme acceptor fragment of ?-galactosidase. The substrate finds application for the sensitive detection of ?-lactamase for direct detection of the enzyme or when the enzyme is used as a label.

Abstract: Truncated fragments of the small fragment of ?-galactosidase are provided that have low affinity for the large fragment of ?-galactosidase and provide for robust signals when two fusion proteins are complexed due to the binding of the proteins to which the ?-galactosidase fragments are fused. The truncated fragments do not interfere with the complexing of the two proteins and allow for the two proteins to function and be responsive to candidate compounds that affect complex formation.

Abstract: A system is provided an enzyme donor (“ED”) fused a surrogate of a mammalian protein of interest, where the fusion protein has the function of the natural protein. A vector is provided comprising a regulatory region functional in a mammalian host cell, a sequence encoding the ED joined to a multiple cloning site, an enzyme acceptor (“EA”) protein or enzyme acceptor sequence encoding such protein, and substrate for the enzyme formed by ED and EA.

Abstract: Methods and reagents are provided for measuring protease activity. The reagent comprises a surface to which is linked an enzyme donor fragment through a protease recognition sequence, where the enzyme donor fragment complexes with an enzyme acceptor fragment to form an active indicator enzyme when the enzyme donor fragment is cleaved from said surface. Conveniently, the surface is a cell membrane surface where the reagent is expressed in the cell. The method comprises bringing together the reagent, the protease, the enzyme acceptor and substrate for the indicator enzyme and measuring the indicator enzyme activity as a measure of the protease activity. The method finds application in screening for compounds modulating the activity of the protease.

Abstract: Methods and compositions are provided for determining ADP in the presence of ATP. These comprise including among the assay reagents at least one of the correcting components creatine phosphokinase and phosphocreatine, pyruvate kinase and phosphoenolpyruvate, peroxidase and a non-interfering peroxidase substrate, and catalase. One aspect of the method employs formation of hydrogen peroxide from the ADP by pyruvate kinase, phosphoenolpyruvate and pyruvate oxidase. The hydrogen peroxide is then determined. A combined reagent having all of the reagents may optionally include a peroxidase when the hydrogen peroxide is to be enzymatically determined. A peroxidase substrate is added to the sample in conjunction with the peroxidase substrate reagent, the mixture incubated and depending on whether the peroxidase substrate is a fluorescer or chemiluminescer, the mixture may be illuminated with excitation light and the emitted light determined as a measure of the ADP in the sample.

Abstract: A sensitive intracellular calcium assay is disclosed comprising conveniently a reagent comprised of a dye precursor capable of entering cells and being hydrolyzed to a dye, whereby the dye complexes with calcium in the cells and provides a luminescent signal, an antibody specific for the dye and conjugated with a quencher, and a cellular anion exchange enzyme inhibitor. In performing the assay, the reagent is combined with cells expressing a receptor responsive to a ligand resulting in a change in cytosolic calcium. After incubation for the dye precursor to permeate the cells, the calcium may be determined by exciting the dye precursor and determining the peak fluorescence over a time course. The method can be used for measuring the effect of an agent on cytosolic calcium by binding to a cell surface membrane receptor.

Abstract: A protein reagent is provided for measuring protease enzyme activity in a sample. The protein reagent comprises two inhibition entities joined by a linker comprised of an indicator enzyme donor and an amino acid sequence susceptible to enzymatic cleavage. The protein reagent is substantially inhibited from binding to the cognate enzyme acceptor fragment, while the product of the enzymatic cleavage binds to the cognate enzyme acceptor fragment to form a functional indicator enzyme. The indicator enzyme activity is related to the protease enzyme activity of the sample.