Method for isolating subpopulations of proteins that engage in protein-protein interactions

Abstract

The invention provides a method for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a chemically reactive supporting matrix to isolate proteins that in turn non-covalently bind other proteins. The supporting matrix is isolated, and the non-covalently bound proteins are subsequently released for analysis. Because the proteins are accessible to chemical manipulation at both the binding and release steps, identification of the non-covalently bound proteins yields information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The method has the advantage of screening the entire proteome simultaneously, unlike two-hybrid systems or phage display methods which can only detect proteins binding to a single bait protein at a time. The method is applicable to the study of protein-protein interactions in biopsy and autopsy specimens, to the study of protein-protein interactions in the presence of signalling molecules, pharmacological agents or toxins, and for comparison of diseased and normal tissues or cancerous and untransformed cells.

Description

field of invention [0001] The invention relates to the study of protein-protein interactions and is expected to find application in the fields of biochemical signal transduction, proteomics, drug discovery, toxicology and diagnostics. Background of the invention [0002] Protein-protein interactions are fundamental to a variety of physiological processes. Cellular processes such as neuronal signaling, cell development, growth and replication all depend on complex networks of protein-protein and protein-small molecule interactions in the cell. These interactions can be classified as constitutive interactions such as between hemoglobin subunits and signal-dependent interactions such as between cAMP-dependent protein kinase subunits or GTP-binding protein subunits. The complexity of the task of studying these interactions is evident from the potential number of protein interactions: a full screen of 15,000 proteins for binary interactions requires te...

AI Technical Summary

Problems solved by technology

Therefore, native protein interactions or interactions resulting from signaling related to physiological processes such as learning or Alzheimer's disease cannot be studied with this technique

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