Protein Complementation Assay detection

Fig. 4.1-7 Protein complementation assays. A protein that carries out a detectable function is separated into two fragments that show no detectable...

Rebois, R.V., Robitaille, M., Petrin, D., Zylbergold, P., Trieu, P., and Hebert, T.E. (2008) Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells. Methods (San Diego, Calif), 45, 214-218. 

Detecting interactions by protein fragment complementation assays... 

Since the initial paper by Fields and Song, there have been significant technical improvements in the method. DNA-binding domains and transcription activation domains have been optimized to reduce false positives and increase the transcription read-out. A variety of reporter plasmids have been engineered to detect a broad range of protein-protein interactions. Much more is understood about the nature of false positives and how to rout them out. Moreover, in response to the utility of this approach, several laboratories have begun to develop transcription-based assays that can be carried out in bacteria, or protein-protein interaction assays based on alternate readouts such as enzyme complementation or fluorescence resonance energy transfer (FRET). 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Remy 1, Campbell-Valois FX, Michnick SW. Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc. 2007 2 2120-2125. 

Rossi F, Charlton CA, Blau HM. Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc. Natl. Acad. Sci. U.S.A. 1997 94 8405-8410. Wehrman TS, Casipit CL, Gewertz NM, Blau HM. Enzymatic detection of protein translocation. Nat. Methods 2005 2 521-527. Hammer MM, Wehrman TS, Blau HM. A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization. FASEB J. 2007 21 3827-3834. 

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