Journal of Structural and Functional Genomics

, Volume 16, Issue 3–4, pp 113–128 | Cite as

Gene selection and cloning approaches for co-expression and production of recombinant protein–protein complexes

  • György Babnigg
  • Robert Jedrzejczak
  • Boguslaw Nocek
  • Adam Stein
  • William Eschenfeldt
  • Lucy Stols
  • Norman Marshall
  • Alicia Weger
  • Ruiying Wu
  • Mark Donnelly
  • Andrzej Joachimiak


Multiprotein complexes play essential roles in all cells and X-ray crystallography can provide unparalleled insight into their structure and function. Many of these complexes are believed to be sufficiently stable for structural biology studies, but the production of protein–protein complexes using recombinant technologies is still labor-intensive. We have explored several strategies for the identification and cloning of heterodimers and heterotrimers that are compatible with the high-throughput (HTP) structural biology pipeline developed for single proteins. Two approaches are presented and compared which resulted in co-expression of paired genes from a single expression vector. Native operons encoding predicted interacting proteins were selected from a repertoire of genomes, and cloned directly to expression vector. In an alternative approach, Helicobacter pylori proteins predicted to interact strongly were cloned, each associated with translational control elements, then linked into an artificial operon. Proteins were then expressed and purified by standard HTP protocols, resulting to date in the structure determination of two H. pylori complexes.


Multiprotein complexes High-throughput structural biology Ligation independent cloning Multigene expression 



The authors wish to thank members of the Structural Biology Center at Argonne National Laboratory for their help with data collection at the 19-ID beamline. This work was supported by the National Institutes of Health Grants GM074942 and GM094585, and by the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Author contributions

GB, R.J., M.D. and A.J. designed the experiments. R.J. and W.E. performed the cloning and small scale expression of the ‘Eps-RBS-fusion-strategy’ and ‘Operon-strategy’ targets. L.S., N.M., A.W. and R.W. purified and crystallized complexes, B.N and A.S. determined the structure of the protein complexes. All authors read and revised the manuscript.

Supplementary material

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Fig.S1 Supplementary material 1 (TIFF 702 kb)
10969_2015_9200_MOESM2_ESM.tif (359 kb)
Fig.S2 Supplementary material 2 (TIFF 358 kb)
10969_2015_9200_MOESM3_ESM.tif (1 mb)
Fig.S3 Supplementary material 3 (TIFF 1036 kb)


  1. 1.
    Alexandrov A, Vignali M, LaCount DJ, Quartley E, de Vries C, De Rosa D, Babulski J, Mitchell SF, Schoenfeld LW, Fields S, Hol WG, Dumont ME, Phizicky EM, Grayhack EJ (2004) A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics 3(9):934–938PubMedCrossRefGoogle Scholar
  2. 2.
    Alm EJ, Huang KH, Price MN, Koche RP, Keller K, Dubchak IL, Arkin AP (2005) The MicrobesOnline Web site for comparative genomics. Genome Res 15(7):1015–1022PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Aziz RK, Devoid S, Disz T, Edwards RA, Henry CS, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Stevens RL, Vonstein V, Xia F (2012) SEED servers: high-performance access to the SEED genomes, annotations, and metabolic models. PLoS One 7(10):e48053PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Babnigg G, Giometti CS (2006) A database of unique protein sequence identifiers for proteome studies. Proteomics 6(16):4514–4522PubMedCrossRefGoogle Scholar
  5. 5.
    Bieniossek C, Nie Y, Frey D, Olieric N, Schaffitzel C, Collinson I, Romier C, Berger P, Richmond TJ, Steinmetz MO, Berger I (2009) Automated unrestricted multigene recombineering for multiprotein complex production. Nat Methods 6(6):447–450PubMedCrossRefGoogle Scholar
  6. 6.
    Bowers PM, Pellegrini M, Thompson MJ, Fierro J, Yeates TO, Eisenberg D (2004) Prolinks: a database of protein functional linkages derived from coevolution. Genome Biol 5(5):R35PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Chaban Y, Boekema EJ, Dudkina NV (2014) Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation. Biochim Biophys Acta 1837(4):418–426PubMedCrossRefGoogle Scholar
  8. 8.
    Cormier CY, Mohr SE, Zuo D, Hu Y, Rolfs A, Kramer J, Taycher E, Kelley F, Fiacco M, Turnbull G, LaBaer J (2010) Protein structure initiative material repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res 38(Database issue):D743–D749PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Corthesy-Theulaz IE, Bergonzelli GE, Henry H, Bachmann D, Schorderet DF, Blum AL, Ornston LN (1997) Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family. J Biol Chem 272(41):25659–25667PubMedCrossRefGoogle Scholar
  10. 10.
    Dehal PS, Joachimiak MP, Price MN, Bates JT, Baumohl JK, Chivian D, Friedland GD, Huang KH, Keller K, Novichkov PS, Dubchak IL, Alm EJ, Arkin AP (2010) MicrobesOnline: an integrated portal for comparative and functional genomics. Nucleic Acids Res 38(Database issue):D396–D400PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Dieckman L, Gu M, Stols L, Donnelly MI, Collart FR (2002) High throughput methods for gene cloning and expression. Protein Expr Purif 25(1):1–7PubMedCrossRefGoogle Scholar
  12. 12.
    Donnelly MI, Zhou M, Millard CS, Clancy S, Stols L, Eschenfeldt WH, Collart FR, Joachimiak A (2006) An expression vector tailored for large-scale, high-throughput purification of recombinant proteins. Protein Expr Purif 47(2):446–454PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132PubMedCrossRefGoogle Scholar
  14. 14.
    Enright AJ, Iliopoulos I, Kyrpides NC, Ouzounis CA (1999) Protein interaction maps for complete genomes based on gene fusion events. Nature 402(6757):86–90PubMedCrossRefGoogle Scholar
  15. 15.
    Ermolaeva MD, White O, Salzberg SL (2001) Prediction of operons in microbial genomes. Nucleic Acids Res 29(5):1216–1221PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Eschenfeldt WH, Stols L, Millard CS, Joachimiak A, Donnelly MI (2009) A family of LIC vectors for high-throughput cloning and purification of proteins. Methods Mol Biol 498:105–115PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Falguieres T, Luyet PP, Gruenberg J (2009) Molecular assemblies and membrane domains in multivesicular endosome dynamics. Exp Cell Res 315(9):1567–1573PubMedCrossRefGoogle Scholar
  18. 18.
    Frydman J, Hartl FU (1996) Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms. Science 272(5267):1497–1502PubMedCrossRefGoogle Scholar
  19. 19.
    Gavin AC, Aloy P, Grandi P, Krause R, Boesche M, Marzioch M, Rau C, Jensen LJ, Bastuck S, Dumpelfeld B, Edelmann A, Heurtier MA, Hoffman V, Hoefert C, Klein K, Hudak M, Michon AM, Schelder M, Schirle M, Remor M, Rudi T, Hooper S, Bauer A, Bouwmeester T, Casari G, Drewes G, Neubauer G, Rick JM, Kuster B, Bork P, Russell RB, Superti-Furga G (2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440(7084):631–636PubMedCrossRefGoogle Scholar
  20. 20.
    Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, Smith HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215–1220PubMedCrossRefGoogle Scholar
  21. 21.
    Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182(2):319–326PubMedCrossRefGoogle Scholar
  22. 22.
    Guell M, van Noort V, Yus E, Chen WH, Leigh-Bell J, Michalodimitrakis K, Yamada T, Arumugam M, Doerks T, Kuhner S, Rode M, Suyama M, Schmidt S, Gavin AC, Bork P, Serrano L (2009) Transcriptome complexity in a genome-reduced bacterium. Science 326(5957):1268–1271PubMedCrossRefGoogle Scholar
  23. 23.
    Ha NC, Oh ST, Sung JY, Cha KA, Lee MH, Oh BH (2001) Supramolecular assembly and acid resistance of Helicobacter pylori urease. Nat Struct Biol 8(6):505–509PubMedCrossRefGoogle Scholar
  24. 24.
    Haffke M, Marek M, Pelosse M, Diebold ML, Schlattner U, Berger I, Romier C (2015) Characterization and production of protein complexes by co-expression in Escherichia coli. Methods Mol Biol 1261:63–89PubMedCrossRefGoogle Scholar
  25. 25.
    Haffke M, Viola C, Nie Y, Berger I (2013) Tandem recombineering by SLIC cloning and Cre-LoxP fusion to generate multigene expression constructs for protein complex research. Methods Mol Biol 1073:131–140PubMedCrossRefGoogle Scholar
  26. 26.
    Huynen MA, Bork P (1998) Measuring genome evolution. Proc Natl Acad Sci USA 95(11):5849–5856PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Jackson RJ, Hellen CU, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11(2):113–127PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Janga SC, Babu MM (2009) Transcript stability in the protein interaction network of Escherichia coli. Mol BioSyst 5(2):154–162PubMedCrossRefGoogle Scholar
  29. 29.
    Joachimiak A (2009) High-throughput crystallography for structural genomics. Curr Opin Struct Biol 19(5):573–584PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Kall L, Krogh A, Sonnhammer EL (2007) Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server. Nucleic Acids Res 35(Web Server issue):W429–W432PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Karp PD, Riley M, Saier M, Paulsen IT, Collado-Vides J, Paley SM, Pellegrini-Toole A, Bonavides C, Gama-Castro S (2002) The EcoCyc database. Nucleic Acids Res 30(1):56–58PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Kather B, Stingl K, van der Rest ME, Altendorf K, Molenaar D (2000) Another unusual type of citric acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase. J Bacteriol 182(11):3204–3209PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Kawai M, Furuta Y, Yahara K, Tsuru T, Oshima K, Handa N, Takahashi N, Yoshida M, Azuma T, Hattori M, Uchiyama I, Kobayashi I (2011) Evolution in an oncogenic bacterial species with extreme genome plasticity: Helicobacter pylori East Asian genomes. BMC Microbiol 11:104PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Kim Y, Babnigg G, Jedrzejczak R, Eschenfeldt WH, Li H, Maltseva N, Hatzos-Skintges C, Gu M, Makowska-Grzyska M, Wu R, An H, Chhor G, Joachimiak A (2011) High-throughput protein purification and quality assessment for crystallization. Methods 55(1):12–28PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Kuhner S, van Noort V, Betts MJ, Leo-Macias A, Batisse C, Rode M, Yamada T, Maier T, Bader S, Beltran-Alvarez P, Castano-Diez D, Chen WH, Devos D, Guell M, Norambuena T, Racke I, Rybin V, Schmidt A, Yus E, Aebersold R, Herrmann R, Bottcher B, Frangakis AS, Russell RB, Serrano L, Bork P, Gavin AC (2009) Proteome organization in a genome-reduced bacterium. Science 326(5957):1235–1240PubMedCrossRefGoogle Scholar
  36. 36.
    Latchman DS (1997) Transcription factors: an overview. Int J Biochem Cell Biol 29(12):1305–1312PubMedCrossRefGoogle Scholar
  37. 37.
    Li Z, Srivastava P (2004) Heat-shock proteins. Curr Protoc Immunol Appendix 1:Appendix 1TGoogle Scholar
  38. 38.
    Makowska-Grzyska M, Kim Y, Maltseva N, Li H, Zhou M, Joachimiak G, Babnigg G, Joachimiak A (2014) Protein production for structural genomics using E. coli expression. Methods Mol Biol 1140:89–105PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Malecki M, Jedrzejczak R, Puchta O, Stepien PP, Golik P (2008) In vivo and in vitro approaches for studying the yeast mitochondrial RNA degradosome complex. Methods Enzymol 447:463–488PubMedCrossRefGoogle Scholar
  40. 40.
    Malecki M, Jedrzejczak R, Stepien PP, Golik P (2007) In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence. J Mol Biol 372(1):23–36PubMedCrossRefGoogle Scholar
  41. 41.
    Merino E, Jensen RA, Yanofsky C (2008) Evolution of bacterial trp operons and their regulation. Curr Opin Microbiol 11(2):78–86PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr 62(Pt 8):859–866PubMedCrossRefGoogle Scholar
  43. 43.
    Moreno-Hagelsieb G, Collado-Vides J (2002) A powerful non-homology method for the prediction of operons in prokaryotes. Bioinformatics 18(Suppl 1):S329–S336PubMedCrossRefGoogle Scholar
  44. 44.
    Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53(Pt 3):240–255PubMedCrossRefGoogle Scholar
  45. 45.
    Nusca TD, Kim Y, Maltseva N, Lee JY, Eschenfeldt W, Stols L, Schofield MM, Scaglione JB, Dixon SD, Oves-Costales D, Challis GL, Hanna PC, Pfleger BF, Joachimiak A, Sherman DH (2012) Functional and structural analysis of the siderophore synthetase AsbB through reconstitution of the petrobactin biosynthetic pathway from Bacillus anthracis. J Biol Chem 287(19):16058–16072PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Olins PO, Rangwala SH (1989) A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem 264(29):16973–16976PubMedGoogle Scholar
  47. 47.
    Osbourn AE, Field B (2009) Operons. Cell Mol Life Sci 66(23):3755–3775PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Overbeek R, Fonstein M, D’Souza M, Pusch GD, Maltsev N (1999) The use of gene clusters to infer functional coupling. Proc Natl Acad Sci USA 96(6):2896–2901PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO (1999) Assigning protein functions by comparative genome analysis: protein phylogenetic profiles. Proc Natl Acad Sci USA 96(8):4285–4288PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Perrakis A, Romier C (2008) Assembly of protein complexes by coexpression in prokaryotic and eukaryotic hosts: an overview. Methods Mol Biol (Clifton, NJ) 426:247–256CrossRefGoogle Scholar
  51. 51.
    Pfleger BF, Kim Y, Nusca TD, Maltseva N, Lee JY, Rath CM, Scaglione JB, Janes BK, Anderson EC, Bergman NH, Hanna PC, Joachimiak A, Sherman DH (2008) Structural and functional analysis of AsbF: origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis. Proc Natl Acad Sci USA 105(44):17133–17138PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Price MN, Arkin AP, Alm EJ (2006) The life-cycle of operons. PLoS Genet 2(6):e96PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Price MN, Huang KH, Alm EJ, Arkin AP (2005) A novel method for accurate operon predictions in all sequenced prokaryotes. Nucleic Acids Res 33(3):880–892PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Prudencio M, Ubbink M (2004) Transient complexes of redox proteins: structural and dynamic details from NMR studies. J Mol Recognit 17(6):524–539PubMedCrossRefGoogle Scholar
  55. 55.
    Pyndiah S, Lasserre JP, Menard A, Claverol S, Prouzet-Mauleon V, Megraud F, Zerbib F, Bonneu M (2007) Two-dimensional blue native/SDS gel electrophoresis of multiprotein complexes from Helicobacter pylori. Mol Cell Proteomics 6(2):193–206PubMedCrossRefGoogle Scholar
  56. 56.
    Qi Y, Balem F, Faloutsos C, Klein-Seetharaman J, Bar-Joseph Z (2008) Protein complex identification by supervised graph local clustering. Bioinformatics 24(13):i250–i258PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Qi Y, Bar-Joseph Z, Klein-Seetharaman J (2006) Evaluation of different biological data and computational classification methods for use in protein interaction prediction. Proteins 63(3):490–500PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Qi Y, Klein-Seetharaman J, Bar-Joseph Z (2007) A mixture of feature experts approach for protein–protein interaction prediction. BMC Bioinformatics 8(Suppl 10):S6PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J, Schachter V, Chemama Y, Labigne A, Legrain P (2001) The protein–protein interaction map of Helicobacter pylori. Nature 409(6817):211–215PubMedCrossRefGoogle Scholar
  60. 60.
    Razick S, Magklaras G, Donaldson IM (2008) iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics 9:405PubMedCentralPubMedCrossRefGoogle Scholar
  61. 61.
    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP, Vidal M (2005) Towards a proteome-scale map of the human protein–protein interaction network. Nature 437(7062):1173–1178PubMedCrossRefGoogle Scholar
  62. 62.
    Selleck W, Tan S (2008) Recombinant protein complex expression in E. coli. Curr Protoc Protein Sci Chapter 5:Unit 5 21Google Scholar
  63. 63.
    Shi R, Munger C, Asinas A, Benoit SL, Miller E, Matte A, Maier RJ, Cygler M (2010) Crystal structures of apo and metal-bound forms of the UreE protein from Helicobacter pylori: role of multiple metal binding sites. Biochemistry 49(33):7080–7088PubMedCrossRefGoogle Scholar
  64. 64.
    Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI (2002) A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr Purif 25(1):8–15PubMedCrossRefGoogle Scholar
  65. 65.
    Stols L, Zhou M, Eschenfeldt WH, Millard CS, Abdullah J, Collart FR, Kim Y, Donnelly MI (2007) New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols. Protein Expr Purif 53(2):396–403PubMedCrossRefGoogle Scholar
  66. 66.
    Tan S (2001) A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein Expr Purif 21(1):224–234PubMedCrossRefGoogle Scholar
  67. 67.
    Timsit Y, Acosta Z, Allemand F, Chiaruttini C, Springer M (2009) The role of disordered ribosomal protein extensions in the early steps of eubacterial 50 s ribosomal subunit assembly. Int J Mol Sci 10(3):817–834PubMedCentralPubMedCrossRefGoogle Scholar
  68. 68.
    Ubbink M (2009) The courtship of proteins: understanding the encounter complex. FEBS Lett 583(7):1060–1066PubMedCrossRefGoogle Scholar
  69. 69.
    Vos MJ, Hageman J, Carra S, Kampinga HH (2008) Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families. Biochemistry 47(27):7001–7011PubMedCrossRefGoogle Scholar
  70. 70.
    Wojcik J, Schachter V (2001) Protein–protein interaction map inference using interacting domain profile pairs. Bioinformatics 17(Suppl 1):S296–S305PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht (outside the USA) 2015

Authors and Affiliations

  • György Babnigg
    • 1
  • Robert Jedrzejczak
    • 1
  • Boguslaw Nocek
    • 1
  • Adam Stein
    • 1
  • William Eschenfeldt
    • 1
  • Lucy Stols
    • 1
  • Norman Marshall
    • 1
  • Alicia Weger
    • 1
  • Ruiying Wu
    • 1
  • Mark Donnelly
    • 1
  • Andrzej Joachimiak
    • 1
  1. 1.Midwest Center for Structural Genomics, Biosciences DivisionArgonne National LaboratoryArgonneUSA

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