Protein Complementation Assay

The mDHFR protein complementation assay has been used to map a signal transduction network that controls the initiation of translation in eukaryotes (Remy and Michnick, 2001). A total of 35 different pairs of full-length proteins were analyzed and 14 interactions were identified using the survival selection of cells grown in the absence of nucleotides. In addition, the use of the fMTX reagent in combination with fluorescence microscopy was used to localize the protein complex within cells (Remy and Michnick, 2001). 

Figure 5.10. Protein complementation assay using murine DHFR. The F[l,2] and F[3] fragments are each fused to the homodimerizing GCN4 leucine zipper protein. A. Transformation of both Z-F[l,2] and Z-F[3] constructs results in reconstituted DHFR and growth of E. coh on agar plates containing trimethoprim. B. Transformation of Z-F[l,2] or Z-F[3] alone does not result in trimethoprim resistant E. coli cells. Figure adapted from Pelletier et al. (1998).

Tab. 7.2. Protein complementation assays used to perform two hybrid assays...

For a laboratory new to the three-hybrid assay, we recommend beginning with the yeast two-hybrid system, which is based on reconstitution of a eukaryotic transcriptional activator protein. Not only is this assay straightforward to practice but also all the necessary strains and plasmids are commercially available. As discussed below, however, there are potential advantages to working in E. coli or using a nontranscription-based assay. Several E. coli-based transcription assays and general protein complementation assays (PCA) have now been developed as two-hybrid assays. Notably, while the E. coli transcription assays have proven amenable to the introduction of small molecule CIDs, the PCAs have not. 

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

Luker, G., Luker, K. (2011). Luciferase protein complementation assays for bioluminescence imaging of cells and mice. Methods in Molecular Biology, 680, 29-43. 

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... 

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

The fl-galactosidase complementation assay has also been adapted for use in mammalian cells (Rossi et al., 1997). The availability of fluorescent substrates for (3-galactosidase allows for fluorescence microscopy and FACS analysis of mammalian cells expressing the fusion proteins of interest. Therefore, similar to the mDHFR system, fl-galactosidase complementation assays may prove useful for genome-scale studies of protein-protein interactions in mammalian cells. 

Remy, I., and Michnick, S. W. (1999). Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays. Proc. Natl. Acad. Sci. USA 96, 5394-5399. 

Systematic Analysis of Complex Signal Transduction Pathways Using Protein Fragment Complementation Assays... 

Michnick SW, Ear PH, Manderson EN et al (2007) Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat Rev Drug Discov 6 569-582... 

Examples of such protein-protein interaction selection systems are phage display (Smith, 1985 Winter et al., 1994), display on other viruses (Kasahara et al., 1994), bacterial surface display (Georgiou et al., 1993 Daugherty et al., 1999), yeast display (Kieke et al., 1997 Boder and Wittrup, 1997), the yeast two hybrid system (Fields and Song, 1989 Chein et al., 1991), and protein-fragment complementation assays (Pelletier et al., 1998). These methods all contain a necessary in vivo step, which has a number of disadvantages that will be discussed in the following sections. 

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. 

Yu H, West M, Keon BH, Bilter GK, Owens S, Lamerdin J, Westwick JK. Measuring drug action in the cellular context using protein-fragment complementation assays. Assay Drug Dev. Technol. 2003, 1, 811-822. 

De A, Gambhir SS. Noninvasive imaging of protein-protein interactions from live cells and living snbjects nsing biolnminescence resonance energy transfer. Faseb J 2005 19 2017-2019. Michnick SW, Ear PH, Manderson EN, Remy I, Stefan E. Universal strategies in research and dmg discovery based on protein-fragment complementation assays. Nat. Rev. Dmg Dis-cov. 2007 6 569-582. 

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