Current methods of detecting interactions of binding pair partners by activation of a responder protein rely primarily on the complementation of inactive responder fragments or subunits to reconstitute the active responder. Typically, molecules of interest, e.g., members of binding pairs, are fused to the fragments or subunits, which do not complement on their own, but can do so when brought together by interaction of the binding pair members. For example, the yeast two-hybrid system has been used to identify cDNA translation products that interact with a protein of interest. This system uses fragments of a gene transcription factor fused to the protein of interest and candidate cDNA products, such that when an interaction occurs inside yeast cells, the fragments complement to activate expression of a responder gene. Another system, based on complementation of fragments of the enzyme dihydrofolate reductase (DHFR), has been used to monitor the interaction of proteins both inside and on the surface of mammalian cells using a fluorescent inhibitor of the enzyme to detect interactions stoichiometrically. Fragment complementation systems of the enzyme β-lactamase have also been used for detection of protein-protein interactions in mammalian cells using a fluorogenic substrate for signal amplification, and in the periplasmic space of gram-negative bacteria using enzyme-conferred antibiotic resistance to select interacting pairs (see, e.g., co-pending U.S. patent application Ser. No. 09/526,106). Similarly, low-affinity truncations of subunits of the enzyme β-galactosidase have been used to monitor protein-protein interactions both inside and on the surface of mammalian cells using a fluorogenic substrate of the enzyme.
In spite of their utility, all of the forgoing systems have drawbacks related to two fundamental properties of such systems. First, unnatural fragments of natural proteins tend to be inherently unstable because they necessarily expose hydrophobic structures, which are normally sequestered in the interior of the protein, to the aqueous environment. Likewise, mutant subunits tend to be unstable when assembly of the complex is delayed or prevented. As a result, the usefulness of such systems for the discovery of natural interactors, for example, in libraries of expressed sequences or of antigen-specific antibodies in repertoire libraries, is seriously compromised because many of the fusions comprising the unstable fragments or subunits may not be stable enough to facilitate detectable interactions. Furthermore, the inherent instability of the components makes them poorly suited for therapeutic use or for in vitro applications, such as clinical diagnostics.
The second compromising property of the foregoing systems is that natural proteins reconstituted from fragments or low-affinity mutant subunits typically have specific activities that are orders of magnitude lower than those of the intact protein. Insofar as the specific activity of the responder is the principal determinant of the sensitivity of the system, the latter will be similarly affected.
The current invention circumvents many of these limitations by using intact, natural proteins as responders, inhibitors, and reactivators. Thus, the full activity of the responder is available for more sensitive detection of the molecule or interaction of interest, and the stable components make these systems suitable for many applications for which fragment or subunit complementation systems are not practical. For example, analyte-activated systems in which responder activation is directly coupled to interaction with a target analyte can form the basis of sensitive and convenient analyte assays. Such assays are homogeneous, requiring no manipulations other than mixing a clinical specimen with the components of a system of the invention, which include responder, inhibitor, and reactivator fused to molecules that bind the analyte, and in so doing shift the equilibrium of inhibitor binding from the responder to the reactivator, thereby activating the responder, such that the responder output is directly proportional to the absolute amount of analyte in the specimen.