Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Systems for the detection and analysis of protein–protein interactions


The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Aronheim A, Zandi E, Hennemann H, Elledge SJ, Karin M (1997) Isolation of an AP-1 repressor by a novel method for detecting protein–protein interactions. Mol Cell Biol 17:3094–3102

  2. Bartel PL, Roecklein JA, SenGuputa D, Fields S (1996) A protein linkage map of Escherichia coli bacteriophage T7. Nat Genet 12:72–77

  3. Berney C, Danuser G (2003) FRET or no FRET: a quantitative comparison. Biophys J 84:3992–4010

  4. Boulton SJ, Gartner A, Reboul J, Vaglio P, Dyson N, Hill DE, Vidal M (2002) Combined functional genomic maps of the C. elegans DNA damage response. Science 295:127–131

  5. Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B, Superti-Furga G (2004) A physical and functional map of the human TNF-α/NF-κB signal transduction pathway. Nat Cell Biol 6:97–105

  6. Broder YC, Katz S, Aronheim A (1998) The ras recruitment system, a novel approach to the study of protein–protein interactions. Curr Biol 8:1121–1124

  7. Chavan M, Yan A, Lennarz WJ (2005) Subunits of the translocon interact with components of the oligosaccharyl transferase complex. J Biol Chem 280:22917–22924

  8. Cooper MA (2003) Label-free screening of bio-molecular interactions. Anal Bioanal Chem 377:834–842

  9. Cooper MA (2004) Advances in membrane receptor screening and analysis. J Mol Recognit 17:286–315

  10. Dang CV, Barrett J, Villa-Garcia M, Resar LM, Kato GJ, Fearon ER (1991) Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization. Mol Cell Biol 11:954–962

  11. Di Guan C, Li P, Riggs PD, Inouye H (1998) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67:21–30

  12. Dmitrova M, Younès-Cauet G, Oertel-Buchheit P, Porte D, Schnarr M, Granger-Schnarr M (1998) A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol Gen Genet 257:205–212

  13. Dove SL, Joung JK, Hochschild A (1997) Activation of prokaryotic transcription through arbitrary protein–protein contacts. Nature 386:627–630

  14. Duplay P, Bedouelle H, Fowler A, Zabin I, Saurin W, Hofnung M (1984) Sequence of malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem 259:10606–10613

  15. Ehrhard KN, Jacoby JJ, Fu XY, Jahn R, Dohlman HG (2000) Use of G-protein fusions to monitor integral membrane protein–protein interactions in yeast. Nat Biotechnol 18:1075–1079

  16. Elangovan M, Wallrabe H, Chen Y, Day RN, Barroso M, Periasamy A (2003) Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29:58–73

  17. Erb EM, Chen X, Allen S, Roberts CJ, Tendler SJB, Davies MC, Forsén S (2000) Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip. Anal Biochem 280:29–35

  18. Evan GI, Lewis GK, Ramsay G, Bishop JM (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616

  19. Ferrando A, Koncz-Kálmán Z, Farràs R, Tiburcio A, Schell J, Koncz C (2001) Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labeling in plant cells. Nucleic Acids Res 29:3685–3693

  20. Fetchko M, Stagljar I (2004) Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions. Methods 32:349–362

  21. Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246

  22. Flajolet M, Rotondo G, Daviet L, Bergametti F, Inchauspé G, Tiollais P, Transy C, Legrain P (2000) A genomic approach of the hepatitis C virus generates a protein interaction map. Gene 242:369–379

  23. Forler D, Köcher T, Rode M, Gentzel M, Izaurralde E, Wilm M (2003) An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat Biotechnol 21:89–92

  24. Galarneau A, Primeau M, Trudeau LE, Michnick SW (2002) β-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein–protein interactions. Nat Biotechnol 20:619–622

  25. Gavin AC, Bösche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Höfert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147

  26. Gilligan JJ, Schuck P, Yergey AL (2002) Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor. Anal Chem 74:2041–2047

  27. Giot L, Bader JS, Brouwer C, Chaudhuri A, Kuang B, Li Y, Hao YL, Ooi CE, Godwin B, Vitols E, Vijayadamodar G, Pochart P, Machineni H, Welsh M, Kong Y, Zerhusen B, Malcolm R, Varrone Z, Collis A, Minto M, Burgess S, McDaniel L, Stimpson E, Spriggs F, Williams J, Neurath K, Ioime N, Agee M, Voss E, Furtak K, Renzulli R, Aanensen N, Carrolla S, Bickelhaupt E, Lazovatsky Y, DaSilva A, Zhong J, Stanyon CA, Finley RL Jr, White KP, Braverman M, Jarvie T, Gold S, Leach M, Knight J, Shimkets RA, McKenna MP, Chant J, Rothberg JM (2003) A protein interaction map of Drosophila melanogaster. Science 320:1727–1736

  28. Guo D, Hazbun TR, Xu XJ, Ng SL, Fields S, Kuo MH (2004) A tethered catalysis, two-hybrid system to identify protein–protein interactions requiring post-translational modifications. Nat Biotechnol 22:888–892

  29. Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P, Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z, Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR, Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J, Poulsen V, Sørensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran MF, Durocher D, Mann M, Hogue CW, Figeys D, Tyers M (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180–183

  30. Hochuli E, Döbeli H, Schacher A (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411:177–184

  31. Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Ceretti DP, Urdal DL, Conlon PJ (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6:1204–1210

  32. Hubsman M, Yudkovsky G, Aronheim A (2001) A novel approach for the identification of protein–protein interaction with integral membrane proteins. Nucleic Acids Res 29:e18

  33. Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA 98:4569–4574

  34. Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG (1991) Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci USA 88:8972–8976

  35. Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395

  36. Johnsson N, Varshavsky A (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci USA 91:10340–10344

  37. Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 95:5752–5756

  38. Karimova G, Dautin N, Ladant D (2005) Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J Bacteriol 187:2233–2243

  39. Karlsson R (2004) SPR for molecular interaction analysis: a review of emerging application areas. J Mol Recog 17:151–161

  40. Karlsson OP, Löfås S (2002) Flow-mediated on-surface recognition of G-protein coupled receptors for applications in surface plasmon resonance biosensors. Anal Biochem 300:132–138

  41. Karlsson R, Michaelsson A, Mattsson L (1991) Kinetic analysis of monoclonal antibody–antigen interactions with a new biosensor based analytical system. J Immunol Methods 145:229–240

  42. Kessler SW (1981) Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cels. Methods Enzymol 73:442–459

  43. Kolmar H, Hennecke F, Götze K, Janzer B, Vogt B, Mayer F, Fritz HJ (1995) Membrane insertion of the bacterial signal transduction protein ToxR and requirements of transcription activation studied by modular replacement of different protein substructures. EMBO J 14:3895–3904

  44. Kondo A, Ueda M (2004) Yeast cell-surface display-applications of molecular display. Appl Microbiol Biotechnol 64:28–40

  45. Kornacker MG, Remsburg B, Menzel R (1998) Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system. Mol Microbiol 30:615–624

  46. Löfås S, Johnsson B (1990) A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. J Chem Soc Chem Commun 21:1526–1528

  47. McCraith S, Holtzman T, Moss B, Fields S (2000) Genome-wide analysis of vaccinia virus protein–protein interactions. Proc Natl Acad Sci USA 97:4879–4884

  48. McDonnell JM (2001) Surface palsmon resonance: towards an understanding of the mechanisms of biological molecular recognition. Curr Opin Chem Biol 5:572–577

  49. Miller JP, Lo RS, Ben-Hur A, Desmarais C, Stagljar I, Noble WS, Fields S (2005) Large-scale identification of yeast integral membrane protein interactions. Proc Natl Acad Sci USA 102:12123–12128

  50. Natsume T, Nakayama H, Jansson O, Isobe T, Takio K, Mikoshiba K (2000) Combination of biomolecular interaction analysis and mass spectrometric amino acid sequencing. Anal Chem 72:4193–4198

  51. Nedelkov D, Nelson RW (2003) Surface plasmon resonance mass spectrometry: recent progress and outlooks. Trends Biotechnol 21:301–305

  52. Nieba L, Nieba-Axmann SE, Persson A, Hämäläinen M, Edebratt F, Hansson A, Lidholm J, Magnusson K, Karlsson AF, Plückthun A (1997) BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem 252:217–228

  53. Obrdlik P, El-Bakkoury M, Hamacher T, Cappellaro C, Vilarino C, Fleischer C, Ellerbrok H, Kamuzinzi R, Ledent V, Blaudez D, Sanders D, Revuelta JL, Boles E, André B, Frommer WB (2004) K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc Natl Acad Sci USA 101:12242–12247

  54. O’Shannessy DJ, Brigham-Burke M, Peck K (1992) Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector. Anal Biochem 205:132–136

  55. Ozawa T, Takeuchi M, Kaihara A, Sato M, Umezawa Y (2001) Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein–protein interactions in bacteria: improved sensitivity and reduced screening time. Anal Chem 73:5866–5874

  56. Paulmurugan R, Gambhir SS (2003) Monitoring protein–protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Anal Chem 75:1584–1589

  57. Pelletier JN, Campbell-Valois FX, Michnick SW (1998) Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc Natl Acad Sci USA 95:12141–12146

  58. Piehler J (2005) New methodologies for measuring protein interactions in vivo and in vitro. Curr Opin Struct Biol 15:4–14

  59. Pollok BA, Heim R (1999) Using GFP in FRET-based applications. Trends Cell Biol 9:57–60

  60. Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J, Schächter V, Chemama Y, Labigne A, Legrain P (2001) The protein–protein interaction map of Helicobacter pylori. Nature 409:211–215

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

  62. Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Séraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032

  63. Rossi F, Charlton CA, Blau HM (1997) Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation. Proc Natl Acad Sci USA 94:8405–8410

  64. Sekar RB, Periasamy A (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160:629–633

  65. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M (1996) Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 93:14440–14445

  66. Shibasaki S, Kuroda K, Duc Nguyen H, Mori T, Zou W, Ueda M. (2005) Detection of protein–protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer. Appl Microbial Biotechnol in press

  67. Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40

  68. Stagljar I, Korostensky C, Johnsson N, te Heesen S (1998) 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

  69. Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H, Wanker EE (2005) A human protein–protein interaction network: a resource for annotating the proteome. Cell 122:957–968

  70. Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846

  71. Suzuki H, Fukunishi Y, Kagawa I, Saito R, Oda H, Endo T, Kondo S, Bono H, Okazaki Y, Hayashizaki Y (2001) Protein–protein interaction panel using mouse full-length cDNAs. Genome Res 11:1758–1765

  72. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533

  73. Ueda M (2004) Future direction of molecular display by yeast-cell surface engineering. J Mol Catal 28:139–143

  74. Ueda M, Tanaka A (2000) Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18:121–140

  75. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM (2000) A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623–627

  76. Vasavada HA, Ganguly S, Germino FJ, Wang ZX, Weissman SM (1991) A contingent replication assay for the detection of protein–protein interactions in animal cells. Proc Natl Acad Sci USA 88:10686–10690

  77. Veraksa A, Bauer A, Artavanis-Tsakonas S (2005) Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry. Dev Dyn 232:827–834

  78. Virella G, Kilpatrick JM, Chenais F, Fudenberg HH (1981) Isolation of soluble immune complexes from human serum: combined use of polyethylene glycol precipitation, gel filtration, and affinity chromatography on protein A-Sepharose. Methods Enzymol 74:644–663

  79. Wallrabe H, Periasamy A (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotechnol 16:19–27

  80. Wallrabe H, Elangovan M, Burchard A, Periasamy A, Barroso M (2003) Conforcal FRET microscopy to measure clustering of ligand–receptor complexes in endocytic membranes. Biophys J 85:559–571

  81. Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287:116–122

  82. Westermarck J, Weiss C, Saffrich R, Kast J, Musti AM, Wessely M, Ansorge W, Séraphin B, Wilm M, Valdez BC, Bohmann D (2002) The DEXD/H-box RNA helicase RHII/Gu is a co-factor for c-Jun-activated transcription. EMBO J 21:451–460

  83. Wilm M, Shevchenko A, Houthaeve T, Breit S, Schweigerer L, Fotsis T, Mann M (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379:466–469

Download references

Author information

Correspondence to Mitsuyoshi Ueda.

Additional information

K. Kuroda, M. Kato and J. Mima contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kuroda, K., Kato, M., Mima, J. et al. Systems for the detection and analysis of protein–protein interactions. Appl Microbiol Biotechnol 71, 127–136 (2006). https://doi.org/10.1007/s00253-006-0395-5

Download citation


  • Surface Plasmon Resonance
  • Fluorescence Resonance Energy Transfer
  • Affinity Resin
  • Surface Plasmon Resonance Biosensor
  • Bait Protein