Abstract: Methods and compositions are provided for determining intracellular translocation of proteins employing ?-galactosidase fragments that independently complex to form an active enzyme. Cells are used having two fusion constructs: one fragment bound to a protein of interest; and the other fragment bound to a compartment localizing signal. The cells are used to screen compounds for their effect on translocation, where a substrate containing high ionic strength solution is used for detection of the enzyme complex.

Abstract: Mitosis of cells is determined, particularly in the presence of a candidate agent, using cells comprising members of an enzyme fragmentation complex pair, where one of the members is in the nucleus and the other member is in the cytoplasm. By growing the cells where mitosis may occur, one adds a substrate providing a detectable product, where the production of the detectable product is indicative of mitosis.

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: Methods and compositions are provided for screening RNAi molecules for efficiency of modulation, particularly inhibition, of expression of genes. The gene for the target protein is fused to a DNA sequence encoding a small fragment of ?-galactosidase. The fragment is competent to complex with a large fragment of ?-galactosidase to form an active enzyme. By adding to any of the fusion protein expressed the large fragment and a substrate that produces a detectable product, the amount of detectable product produced is related to the efficiency of modulation of expression by the RNAi. The method finds particular application in high throughput screening.

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: Methods and compositions are provided for determining cell membrane protein populations in the cell membrane of a cell and changes in the population due to changes in the environment or status of the cell. The methods employ a cell having a fusion construct of the cell membrane protein linked to a signal producing peptide through an exofacial protease recognition site or sites. The signal producing peptide is either an enzyme fragment capable of binding to a second enzyme fragment to form an active enzyme when released from the cell membrane or has two binding sites, where the complementary binding entities are related in that a signal is produced when the two entities are in proximity. For the enzyme signal producing peptide, by adding the protease to the cell and the second enzyme fragment and substrate, one can determine the cell membrane protein population and the effect of changes in the cell environment on such population.

Abstract: A system is provided for producing biologically active fusion proteins comprising a sequence encoding an enzyme donor (“ED”) sequence of fused in reading frame to a sequence encoding 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 transcriptional and translational 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, that is complemented by the ED to form a functional enzyme, e.g. &bgr;-galactosidase, and substrate that is turned over by the enzyme to form a detectable substrate. Mammalian cells are employed that may be modified to provide specific functions, such as expression of the EA, overexpression of a protein of interest, etc. The system is used to monitor the fusion protein as a surrogate for the natural protein.