|Thelen, Jay - UNIV OF MISSOURI|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 18, 2007
Publication Date: February 1, 2008
Citation: Miernyk, J.A., Thelen, J.J. 2008. Biochemical Approaches for Discovering Protein-Protein Interactions. Plant Journal. 53:597-609. Interpretive Summary: None of the components of a cell can function in isolation. Instead, any single component functions in close proximity with other components. The occurrence of multi-component complexes serves to control the function and in some instances the biological lifetime of individual components. Five different methods were developed, and then optimized, for laboratory isolation and study of multi-component complexes from plants. After isolation and purification of the complexes, methods were developed to disrupt interactions between components and identification of the individuals. Multiple strategies were developed, and described, to verify and validate interactions between individual components. This is critically important, because the mis-identification of complexes that do not actually exist in living cells can divert research efforts away from study of legitimate complexes, wasting effort and resources. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.
Technical Abstract: Protein-protein interactions or protein complexes are indigenous to nearly all cellular processes, ranging from metabolism to structure. Elucidating both individual protein associations and complex protein interaction networks, while challenging, is an essential goal of functional genomics. For example, discovering interacting partners for a “protein of unknown function” can provide insight into actual function far beyond what is possible with sequence-based predictions, and provide a platform for future research. Synthetic genetic approaches such as two-hybrid screening often reveal a perplexing array of potential interacting partners for any given target protein. It is now known, however, that this type of anonymous screening approach can yield high levels of false positive results, and therefore putative interactors must be confirmed by independent methods. In vitro biochemical strategies for identifying interacting proteins are varied and time-honored, some as old as the field of protein chemistry itself. Herein we discuss five biochemical approaches for isolating and characterizing protein-protein interactions in vitro; co-immunoprecipitation, blue native gel electrophoresis, in vitro binding assays, protein cross-linking, and rate-zonal centrifugation. A perspective is provided for each method, and where appropriate specific, trial-tested methods and results are included.