1. Field of the Invention
The invention generally relates to methods for isolating and using multi-protein complexes that are biologically active. In particular, the invention provides buffers that may be used to isolate the multi-protein complexes in a biologically active form, and protein arrays that contain biologically active multi-protein complexes that are isolated using such buffers, as well as methods for their use in high throughput screening assays.
2. Background of the Invention
Biotechnology companies are increasingly relying on screening methods and technologies to identify lead compounds with therapeutic capabilities. Such methods typically involve the use of immobilized biologically relevant molecules such as nucleic acids and proteins. The technology for manipulating nucleic acids is well developed due in part to the relative simplicity of DNA and RNA molecules and the hybridization reactions they undergo. In contrast, the development of protein arrays is much more challenging due to the complexity of protein-ligand interactions.
Several factors contribute to this complexity. The 20 (common) amino acids that make up proteins are each unique and possess widely differing chemical properties. Thus, individual proteins, each with their own characteristic amino acid composition, also differ widely from one another, and exhibit widely differing biological activities. Further, in addition to linear primary structure, native “folded” proteins possess characteristic secondary (and sometimes tertiary and quaternary) 3-dimensional structural elements that are necessary for proper functioning. Maintenance of this native structure depends in part on the surrounding environment (e.g. pH, temperature, hydrophilicity/hydrophobicity of medium, etc.). In addition, in order to carry out its characteristic activity, a protein often must be in contact with and interact with other biological molecules. For example, many proteins require the presence of various cofactors, activators, and/or other proteins (e.g. scaffolding proteins) in order to be active. In vitro reproduction of the in vivo-like conditions necessary for proper protein functioning can thus be extremely challenging.
Nevertheless, a large number of protein array-based products for a range of applications have been introduced. Progress has been made in developing so-called interaction protein arrays in which a number of proteins (or polypeptides or peptides) are immobilized on a surface and specific interactions with soluble proteins (e.g. in a biological test sample) are detected. Such interactions between immobilized proteins and soluble proteins are intended to mimic biochemical events that take place in a cell or biological fluid in vivo. However, the physiological relevance of data obtained with such biochips is questionable. In a cellular environment, a biochemical reaction will take place only if the reactants are suitably juxtaposed (e.g. in a sub-cellular compartment, on the surface of a cell, etc). Bio-molecules that come together on the surface of a biochip may or may not actually be co-localized in vivo, and, conversely, bio-molecules that come together in vivo may or may not associate properly on the surface of a biochip.
This challenge is particularly acute when dealing with large, multi-protein complexes such as those involved in cell signaling. The assembly of receptors and signal transduction proteins into large multi-protein complexes has emerged as a general mechanism of cellular signaling (see review, Pawson and Scott, 1997), and processes such as phosphorylation, glycosylation, and protein-protein interaction are considered to be major regulators of receptor function. Due to their importance in many cellular processes, methods for isolating and using such complexes are in high demand. Unfortunately, buffers that are routinely used for immunoprecipitation of such protein complexes typically contain sodium sodecyl sulfate (SDS), sodium deoxycholate and/or other detergents which can alter the activity of the receptor itself, or the activity of proteins that surround and interact with the receptor in vivo, such as kinases and phosphatases
The prior art has thus-far failed to provide methods for reliably isolating multi-protein complexes in a biologically active form. The prior art has also thus-far failed to provide arrays of immobilized, biologically active multi-protein complexes in which the in vivo associations between the proteins in the complex are maintained.
USPTO Backgrounds
USPTO Backgrounds