Key Points
-
Protein-fragment complementation assays (PCAs) provide a general methodology to detect and study spatial and temporal dynamics of protein–protein interactions in intact living cells and have many applications, which are listed below.
-
PCAs have been used to engineer novel binding proteins for research and potential therapeutic applications including antibodies and alternative binding proteins.
-
To study mechanisms of membrane receptor-mediated and other signal transduction processes.
-
To probe actions of small molecules on cellular pathways that regulate cell-fate decisions.
-
To predict on- and off-target effects and potential therapeutic applications of small molecules.
-
To link potential disease drug-target genes to specific cellular functions by a general expression-cloning strategy
-
To visualize spatial and temporal dynamics of protein complexes in cultured cells and whole animals.
-
To visualize pharmacodynamics of drugs and effects of drugs on tumour growth in whole animals
Abstract
Changes in the interactions among proteins that participate in a biochemical pathway can reflect the immediate regulatory responses to intrinsic or extrinsic perturbations of the pathway. Thus, methods that allow for the direct detection of the dynamics of protein–protein interactions can be used to probe the effects of any perturbation on any pathway of interest. Here we describe experimental strategies — based on protein-fragment complementation assays (PCAs) — that can achieve this. PCA-based strategies can be used with or instead of traditional target-based drug discovery strategies to identify novel pathway-component proteins of therapeutic interest, to increase the quantity and quality of information about the actions of potential drugs, and to gain insight into the intricate networks that make up the molecular machinery of living cells.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Drews, J. Drug discovery: a historical perspective. Science 287, 1960–1964 (2000).
Chee, M. et al. Accessing genetic information with high-density DNA arrays. Science 274, 610–614 (1996).
Stoughton, R. B. & Friend, S. H. How molecular profiling could revolutionize drug discovery. Nature Rev. Drug Discov. 4, 345–350 (2005). An outstanding review on the applications of microarray profiling to predict on- and off-pathway actions of drugs and genetic manipulation. References 4–6 present interesting examples of such applications.
Marton, M. J. et al. Drug target validation and identification of secondary drug target effects using DNA microarrays. Nature Med. 4, 1293–1301 (1998).
Hardwick, J. S., Kuruvilla, F. G., Tong, J. K., Shamji, A. F. & Schreiber, S. L. Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. Proc. Natl Acad. Sci USA 96, 14866–14870 (1999).
Hughes, T. R. et al. Functional discovery via a compendium of expression profiles. Cell 102, 109–126 (2000).
Giaever, G. et al. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nature Genet. 21, 278–283 (1999). Together with references 8, 9, 13 and 14, illustrate how chemical–gene interactions in yeast can be used to map small molecules to potential protein targets or pathways.
Giaever, G. et al. Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc. Natl Acad. Sci. USA 101, 793–798 (2004).
Hartwell, L. H., Szankasi, P., Roberts, C. J., Murray, A. W. & Friend, S. H. Integrating genetic approaches into the discovery of anticancer drugs. Science 278, 1064–1068 (1997).
Guo, Z., Kumagai, A., Wang, S. X. & Dunphy, W. G. Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev. 14, 2745–2756 (2000).
Kuruvilla, F. G., Shamji, A. F., Sternson, S. M., Hergenrother, P. J. & Schreiber, S. L. Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays. Nature 416, 653–657 (2002).
Bracha-Drori, K. et al. Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419–427 (2004).
Parsons, A. B. et al. Integration of chemical–genetic and genetic interaction data links bioactive compounds to cellular target pathways. Nature Biotech. 22, 62–69 (2004).
Parsons, A. B. et al. Exploring the mode-of-action of bioactive compounds by chemical–genetic profiling in yeast. Cell 126, 611–625 (2006).
Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. From molecular to modular cell biology. Nature 402 (Suppl. 6761), C47–C52. (1999). A concise and thoughtful essay on the meaning of modularity in biological processes.
Tong, A. H. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004). The most comprehensive analysis of gene interactions on a large scale.
Yook, S. H., Oltvai, Z. N. & Barabasi, A. L. Functional and topological characterization of protein interaction networks. Proteomics 4, 928–942 (2004).
Ihmels, J. et al. Revealing modular organization in the yeast transcriptional network. Nature Genet. 31, 370–377 (2002).
Snel, B., Bork, P. & Huynen, M. A. The identification of functional modules from the genomic association of genes. Proc. Natl Acad. Sci. USA 99, 5890–5895 (2002).
Rives, A. W. & Galitski, T. Modular organization of cellular networks. Proc. Natl Acad. Sci. USA 100, 1128–1133 (2003).
Yao, G., Craven, M., Drinkwater, N. & Bradfield, C. A. Interaction networks in yeast define and enumerate the signaling steps of the vertebrate aryl hydrocarbon receptor. PLoS Biol. 2, e65 (2004).
Johnsson, N. & Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl Acad. Sci. USA 91, 10340–10344 (1994). The first description of a detector of protein–protein interactions based on de novo dissection and refolding of a functional protein.
Pelletier, J. N. & Michnick, S. W. A protein complementation assay for detection of protein–protein interactions in vivo. Protein Eng. 10, 89 (1997). Together with references 24 and 25, a first description of the design principles used in creating PCAs.
Pelletier, J. N., Campbell-Valois, F. & Michnick, S. W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl Acad. Sci. USA 95, 12141–12146 (1998).
Michnick, S. W., Remy, I., Campbell-Valois, F. X., Vallee-Belisle, A. & Pelletier, J. N. Detection of protein–protein interactions by protein fragment complementation strategies. Methods Enzymol. 328, 208–230 (2000).
Remy, I. & Michnick, S. W. Clonal selection and in vivo quantitation of protein interactions with protein fragment complementation assays. Proc. Natl Acad. Sci. USA 96, 5394–5399 (1999).
Remy, I., Wilson, I. A. & Michnick, S. W. Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990–993 (1999). Describes the use of a PCA as a 'molecular ruler' and detector of a novel cytokine-receptor activation mechanism.
Remy, I. & Michnick, S. W. Visualization of biochemical networks in living cells. Proc. Natl Acad. Sci. USA 98, 7678–7683 (2001). The first description of the protein–protein interaction sentinel strategy to map the organization and effects of small molecules on signal-transduction pathways. An extension of the approach for identifying small molecule on- and off-pathway effects is described in reference 70.
Galarneau, A., Primeau, M., Trudeau, L. E. & Michnick, S. W. β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nature Biotech. 20, 619–622 (2002).
Spotts, J. M., Dolmetsch, R. E. & Greenberg, M. E. Time-lapse imaging of a dynamic phosphorylation-dependent protein–protein interaction in mammalian cells. Proc. Natl Acad. Sci. USA 99, 15142–15147. (2002). Describes the use of single-cell imaging to quantitate induced dynamic protein–protein interactions by PCA.
Wehrman, T., Kleaveland, B., Her, J. H., Balint, R. F. & Blau, H. M. Protein–protein interactions monitored in mammalian cells via complementation of β-lactamase enzyme fragments. Proc. Natl Acad. Sci. USA 99, 3469–3474 (2002).
Ghosh, I., Hamilton, A. D. & Regan, L. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000).
Hu, C. D., Chinenov, Y. & Kerppola, T. K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798 (2002).
Remy, I. & Michnick, S. A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways. Methods 32, 381–388 (2004). Togther with references 35, 36 and 69, this reference describes the application of PCA as a solution to the generalized expression-cloning problem.
Remy, I. & Michnick, S. W. Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt. Mol. Cell. Biol. 24, 1493–1504 (2004).
Remy, I., Montmarquette, A. & Michnick, S. W. PKB/Akt modulates TGF-β signalling through a direct interaction with Smad3. Nature Cell Biol. 6, 358–365 (2004).
Remy, I. & Michnick, S. W. Mapping biochemical networks with protein-fragment complementation assays. Methods Mol. Biol. 261, 411–426 (2004).
Luker, K. E. et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl Acad. Sci. USA 101, 12288–12293 (2004).
Kaihara, A., Kawai, Y., Sato, M., Ozawa, T. & Umezawa, Y. Locating a protein–protein interaction in living cells via split Renilla luciferase complementation. Anal. Chem. 75, 4176–4181 (2003).
Paulmurugan, R. & Gambhir, S. S. Monitoring protein–protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal. Chem. 75, 1584–1589. (2003).
Remy, I. & Michnick, S. W. A highly sensitive protein–protein interaction assay based on Gaussia luciferase. Nature Methods 3, 977–979 (2006). Together with reference 45, the first report that PCAs can be applied to study the disruption of protein–protein interactions.
Rossi, F., Charlton, C. A. & Blau, H. M. Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation. Proc. Natl Acad. Sci. USA 94, 8405–8410 (1997).
Ozawa, T., Nogami, S., Sato, M., Ohya, Y. & Umezawa, Y. A fluorescent indicator for detecting protein–protein interactions in vivo based on protein splicing. Anal. Chem. 72, 5151–5157 (2000).
Pelletier, J. N., Arndt, K. M., Pluckthun, A. & Michnick, S. W. An in vivo library-versus-library selection of optimized protein–protein interactions. Nature Biotech. 17, 683–690 (1999). Describes the application of a PCA to the directed evolution of pairwise protein–protein interactions.
Stefan, E., Aquin, S., Berger, N. & Michnick, S. W. Quantification of dynamic protein complexes using Renilla luciferase-fragment complementation applied to PKA activities in vivo. Proc. Natl Acad. Sci. USA (in the press).
Magliery, T. J. et al. Detecting protein–protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J. Am. Chem. Soc. 127, 146–157 (2005).
Nyfeler, B., Michnick, S. W. & Hauri, H. P. Capturing protein interactions in the secretory pathway of living cells. Proc. Natl Acad. Sci. USA 102, 6350–6355 (2005). A demonstration that a PCA can capture transient interactions of cargo protein–lectin interactions in secretory pathways. Such interactions are not observable by other techniques.
Arndt, K. M. et al. A heterodimeric coiled–coil peptide pair selected in vivo from a designed library-versus-library ensemble. J. Mol. Biol. 295, 627–639 (2000).
Campbell-Valois, F. X., Tarassov, K. & Michnick, S. W. Massive sequence perturbation of a small protein. Proc. Natl Acad. Sci. USA 102, 14988–14993 (2005). The most dramatic example of an application of a PCA to directed evolution of a protein. The entire natural evolution of a protein was recreated in this experiment.
Campbell-Valois, F. X. & Michnick, S. W. The transition state of the ras binding domain of Raf is structurally polarized based on Phi-values but is energetically diffuse. J. Mol. Biol. 365, 1559–1577 (2007).
Campbell-Valois, F. X., Tarassov, K. & Michnick, S. W. Massive sequence perturbation of the Raf Ras binding domain reveals relationships between sequence conservation, secondary structure propensity, hydrophobic core organization and stability. J. Mol. Biol. 362, 151–171 (2006).
Amstutz, P., Koch, H., Binz, H. K., Deuber, S. A. & Pluckthun, A. Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries. Protein Eng. Des. Sel. 19, 219–229 (2006). This and reference 53 describe elegant applications of PCAs to select for specific binding proteins from naive libraries.
Koch, H., Grafe, N., Schiess, R. & Pluckthun, A. Direct selection of antibodies from complex libraries with the protein fragment complementation assay. J. Mol. Biol. 357, 427–441 (2006).
Cody, V., Luft, J. R., Ciszak, E., Kalman, T. I. & Freisheim, J. H. Crystal structure determination at 2.3 Å of recombinant human dihydrofolate reductase ternary complex with NADPH and methotrexate-γ-tetrazole. Anticancer Drug Design 7, 483–491 (1992).
Aruffo, A. & Seed, B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc. Natl Acad. Sci. USA 84, 8573–8577 (1987). This, together with references 56–58, is a classic example of expression cloning in which cDNAs were identified based on specific protein–protein interactions.
D'Andrea, A. D., Fasman, G. D. & Lodish, H. F. Erythropoietin receptor and interleukin-2 receptor β-chain: a new receptor family. Cell 58, 1023–1024 (1989).
Sako, D. et al. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell 75, 1179–1186 (1993).
Lin, H. Y., Wang, X. F., Ng-Eaton, E., Weinberg, R. A. & Lodish, H. F. Expression cloning of the TGF-β type II receptor, a functional transmembrane serine/threonine kinase. Cell 68, 775–785 (1992).
Grimm, S. The art and design of genetic screens: mammalian culture cells. Nature Rev. Genet. 5, 179–189 (2004). An outstanding review on the expression-cloning problem. See also references 34 and 55.
Pawson, T. & Nash, P. Protein–protein interactions define specificity in signal transduction. Genes Dev. 14, 1027–1047 (2000).
Weston, C. R. & Davis, R. J. Signal transduction: signaling specificity — a complex affair. Science 292, 2439–2440. (2001).
Ito, T. et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl Acad. Sci. USA 98, 4569–4574 (2001).
Ito, T. et al. Toward a protein–protein interaction map of the budding yeast: a comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins. Proc. Natl Acad. Sci. USA 97, 1143–1147. (2000).
Drees, B. L. Progress and variations in two-hybrid and three-hybrid technologies. Curr. Opin. Chem. Biol. 3, 64–70 (1999).
Walhout, A. J. et al. Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287, 116–122 (2000).
Vidal, M. & Legrain, P. Yeast forward and reverse 'n'-hybrid systems. Nucleic Acids Res. 27, 919–929 (1999).
Fields, S. & Song, O. A novel genetic system to detect protein–protein interactions. Nature 340, 245–246 (1989).
Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).
Ding, Z. et al. A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc. Natl Acad. Sci. USA 103, 15014–15019 (2006).
Macdonald, M. L. et al. Identifying off-target effects and hidden phenotypes of drugs in human cells. Nature Chem. Biol. 2, 329–337 (2006). The first detailed study of pathway targeting of small molecules as detected by PCA, PCA and high-content quantitative imaging of protein–protein interactions, and the ability to predict unintended (off-pathway) effects of small molecules.
Paulmurugan, R., Umezawa, Y. & Gambhir, S. S. Noninvasive imaging of protein–protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc. Natl Acad. Sci. USA 99, 15608–15613. (2002). The first report of detecting protein–protein interactions in nude mice injected with cells harbouring a PCA. Imaging of PCA reporters could be applied to analysing pharmacodynamics and effects of anticancer drugs in living subjects. See also references 38, 72–74 and 101.
Paulmurugan, R., Massoud, T. F., Huang, J. & Gambhir, S. S. Molecular imaging of drug-modulated protein–protein interactions in living subjects. Cancer Res. 64, 2113–2119 (2004).
Massoud, T. F., Paulmurugan, R. & Gambhir, S. S. Molecular imaging of homodimeric protein–protein interactions in living subjects. FASEB J. 18, 1105–1107 (2004).
Paulmurugan, R. & Gambhir, S. S. Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal. Chem. 77, 1295–1302 (2005).
Kaletta, T. & Hengartner, M. O. Finding function in novel targets: C. elegans as a model organism. Nature Rev. Drug Discov. 5, 387–398 (2006).
Zhang, S., Ma, C. & Chalfie, M. Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119, 137–144 (2004). A particularly interesting application of the GFP PCA to study the role of pairs of genes in animal development.
Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).
Benton, R., Sachse, S., Michnick, S. W. & Vosshall, L. B. Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol. 4, e20 (2006). Describes the application of a PCA to study membrane topology and secretory processing of an odorant receptor in transgenic flies.
Johnsson, N. & Varshavsky, A. Ubiquitin-assisted dissection of protein transport across membranes. EMBO J. 13, 2686–2698 (1994).
Stagljar, I., Korostensky, C., Johnsson, N. & te Heesen, S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl Acad. Sci. USA 95, 5187–5192. (1998).
Laser, H. et al. A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter. Proc. Natl Acad. Sci. USA 97, 13732–13737 (2000).
Paumi, C. M. et al. Mapping protein–protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis. Mol. Cell 26, 15–25 (2007).
Miller, J. P. et al. Large-scale identification of yeast integral membrane protein interactions. Proc. Natl Acad. Sci. USA 102, 12123–12128 (2005). The first large-scale application of a PCA to identify novel membrane protein–protein interactions in living cells using the ubiquitin split protein sensor. See also reference 82.
Anfinsen, C. B., Haber, E., Sela, M. & White Jr F. H. . The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proc. Natl Acad. Sci. USA 47, 1309–1314 (1961). References 84–88 are must-reads for those interested in the history and implications of protein-fragment complementation to modern theories of protein folding.
Anfinsen, C. B. Principles that govern the folding of protein chains. Science 181, 223–230 (1973).
Gutte, B. & Merrifield, R. B. The synthesis of ribonuclease A. J. Biol. Chem. 246, 1922–1941 (1971).
Richards, F. M. On the enzymatic activity of subtilisin-modified ribonuclease. Proc. Natl Acad. Sci. USA 44, 162–166 (1958).
Taniuchi, H. & Anfinsen, C. B. Simultaneous formation of two alternative enzymology active structures by complementation of two overlapping fragments of staphylococcal nuclease. J. Biol. Chem. 246, 2291–2301 (1971).
Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell. Biol. 3, 906–918. (2002).
Gegg, C. V., Bowers, K. E. & Matthews, C. R. Probing minimal independent folding units in dihydrofolate reductase by molecular dissection. Protein Sci. 6, 1885–1892 (1997).
Remy, I. & Michnick, S. W. Dynamic visualization of expressed gene networks. J. Cell. Physiol. 196, 419–429 (2003).
Subramaniam, R., Desveaux, D., Spickler, C., Michnick, S. W. & Brisson, N. Direct visualization of protein interactions in plant cells. Nature Biotech. 19, 769–772 (2001).
Hu, C. D. & Kerppola, T. K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nature Biotech. 21, 539–545 (2003).
Jach, G., Pesch, M., Richter, K., Frings, S. & Uhrig, J. F. An improved mRFP1 adds red to bimolecular fluorescence complementation. Nature Methods 3, 597–600 (2006).
Mervine, S. M., Yost, E. A., Sabo, J. L., Hynes, T. R. & Berlot, C. H. Analysis of G protein βγ dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of β1 for particular γ subunits. Mol. Pharmacol. 70, 194–205 (2006).
de Virgilio, M., Kiosses, W. B. & Shattil, S. J. Proximal, selective, and dynamic interactions between integrin αIIbβ3 and protein tyrosine kinases in living cells. J. Cell Biol. 165, 305–311 (2004).
Fang, D. & Kerppola, T. K. Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes. Proc. Natl Acad. Sci. USA 101, 14782–14787 (2004).
Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438 (2004).
Grinberg, A. V., Hu, C. D. & Kerppola, T. K. Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol. Cell. Biol. 24, 4294–4308 (2004).
Hynes, T. R., Mervine, S. M., Yost, E. A., Sabo, J. L. & Berlot, C. H. Live cell imaging of Gs and the β2-adrenergic receptor demonstrates that both alphas and β1γ7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the β2-adrenergic receptor. J. Biol. Chem. 279, 44101–44112 (2004).
Paulmurugan, R. & Gambhir, S. S. Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein–protein interactions in cells and living animals. Cancer Res. 65, 7413–7420 (2005).
Phillips, K. J., Rosenbaum, D. M. & Liu, D. R. Binding and stability determinants of the PPARγ nuclear receptor-coactivator interface as revealed by shotgun alanine scanning and in vivo selection. J. Am. Chem. Soc. 128, 11298–11306 (2006).
Tafelmeyer, P., Johnsson, N. & Johnsson, K. Transforming a (β/α)8-barrel enzyme into a split-protein sensor through directed evolution. Chem. Biol. 11, 681–689 (2004).
Paschon, D. E., Patel, Z. S. & Ostermeier, M. Enhanced catalytic efficiency of aminoglycoside phosphotransferase (3′)-IIa achieved through protein fragmentation and reassembly. J. Mol. Biol. 353, 26–37 (2005).
Jullien, N., Sampieri, F., Enjalbert, A. & Herman, J. P. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, e131 (2003).
Thaminy, S., Miller, J. & Stagljar, I. The split-ubiquitin membrane-based yeast two-hybrid system. Methods Mol. Biol. 261, 297–312 (2004).
Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl Acad. Sci. USA 95, 5752–5756 (1998).
Wehr, M. C. et al. Monitoring regulated protein–protein interactions using split TEV. Nature Methods 3, 985–993 (2006).
Acknowledgements
The work published from our group was supported by the CIHR, NSERC, NFSP and NIH. S.W.M. holds the Canada Research Chair in integrative genomics.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
S.W.M. is a shareholder in Odyssey Thera Inc.
Related links
Glossary
- Perturbation
-
Any treatment of a cell, including chemicals, gene deletion or ablation with a short-interfering RNA that may produce an observable effect on an assay that reports activity of a pathway.
- Module
-
A group of genes that are shown to interact with each other to produce a common phenotype, or proteins that are functionally linked and have more physical interactions between themselves than with other proteins.
- Cross-talk
-
Interactions between two pathways that are thought to mediate common or different cellular processes.
- Allosteric
-
A change in the spatial orientation of subunits within a protein complex that is shown to regulate the function of the individual subunits. For example, a change in the affinity of haemoglobin subunits for molecular oxygen on changing from one configuration to another.
- Reversibility
-
In the context of protein-fragment complementation assays, the complete unfolding and separation of protein-reporter fragments when the proteins to which they are attached dissociate.
- Phage-display
-
A method to identify or design proteins with the specific ability to bind to a molecule by expressing proteins as fusions to filamentous bacterial viruses, testing binding to a molecule arrayed on a solid surface, identifying those phage particles that bind and then replicating them in bacteria.
- Expression-cloning
-
The identification of a gene that performs a specific cellular function by expressing a library of cDNAs, and screening for resulting proteins that perform the function.
- Sentinel
-
An assay that provides a direct readout for activity in a specific biochemical pathway.
- Off-pathway effect
-
Effects of a perturbation on pathways that are not predicted to be affected by the perturbation.
- High-content imaging
-
Generally a quantitative analysis of morphological changes or spatial and temporal changes of proteins or protein complexes. Usually performed with fluorescent molecules (for example, antibodies covalently coupled to fluorescent molecules) or fluorescent proteins genetically fused to proteins of interest. These can be imaged by epifluorescence or confocal microscopy.
- Hidden phenotypes
-
A potential outcome to treatment of a cell with some perturbation such as death, differentiation state or morphology that is predicted by pathway sentinel assays, but not necessarily observed in the cells in which the assays were performed.
- Hierarchical clustering
-
A general term for many mathematical strategies used to establish relationships between objects according to common features.
Rights and permissions
About this article
Cite this article
Michnick, S., Ear, P., Manderson, E. et al. Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat Rev Drug Discov 6, 569–582 (2007). https://doi.org/10.1038/nrd2311
Issue Date:
DOI: https://doi.org/10.1038/nrd2311
This article is cited by
-
Functional advantages of building nanosystems using multiple molecular components
Nature Chemistry (2023)
-
Nanomachines built from multiple components can have functional advantages
Nature Chemistry (2023)
-
Tracking mutation and drug-driven alterations of oncokinase conformations
memo - Magazine of European Medical Oncology (2022)
-
Human lysyl-tRNA synthetase evolves a dynamic structure that can be stabilized by forming complex
Cellular and Molecular Life Sciences (2022)
-
Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition
Nature Communications (2020)