Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Protein production and purification

Nature Methods volume 5, pages 135–146 (2008)Cite this article

An Addendum to this article was published on 01 April 2008

This article has been updated

Abstract

NOTE: In the version of this Review initially published, an author (B. Martin Hallberg) was left off of the author list. This information has been added to the HTML and PDF versions of the Review.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Solubility as a function of construct length.
Figure 2: Gel filtration profiles.

Similar content being viewed by others

Change history

  • 04 March 2008

    In the version of this Review initially published, an author (B. Martin Hallberg) was left off of the author list. This information has been added to the HTML and PDF versions of the Review.

References

  1. Sauder, J.M. et al. High throughput protein production and crystallization at NYSGXRC. in Structural Proteomics: High-Throughput Methods Vol. 426 (eds. B. Kobe, M.Guss & H. Thomas) 561–575 (Humana Press, Totowa, New Jersey, USA, 2008).

    Chapter  Google Scholar 

  2. Büssow, K. et al. Structural genomics of human proteins-target selection and generation of a public catalogue of expression clones. Microb. Cell Fact. 4, 21 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Heinemann, U., Büssow, K., Mueller, U. & Umbach, P. Facilities and methods for the high-throughput crystal structural analysis of human proteins. Accounts Chem. Res. 36, 157–163 (2003).

    Article  CAS  Google Scholar 

  4. Banci, L. et al. First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value. Acta Crystallogr. D62, 1208–1217 (2006).

    CAS  Google Scholar 

  5. Aricescu, A.R. et al. Eukaryotic expression: developments for structural proteomics. Acta Crystallogr. D62, 1114–1124 (2006).

    CAS  Google Scholar 

  6. Heinemann, U. Establishing a structural genomics platform: The Berlin-based Protein Structure Factory. Gene Funct. Dis. 3, 25–32 (2002).

    Article  CAS  Google Scholar 

  7. Scheich, C., Kummel, D., Soumailakakis, D., Heinemann, U. & Büssow, K. Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res. 35, e43 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Bartlam, M., Xu, Y. & Rao, Z. Structural proteomics of the SARS coronavirus: a model response to emerging infectious diseases. J. Struct. Funct. Genomics 8, 85–97 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gong, W.M. et al. Structural genomics efforts at the Chinese Academy of Sciences and Peking University. J. Struct. Funct. Genomics 4, 137–139 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Albeck, S. et al. Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC). Acta Crystallogr. D61, 1364–1372 (2005).

    CAS  Google Scholar 

  11. Lesley, S.A. et al. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. USA 99, 11664–11669 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Montelione, G.T., Zheng, D., Huang, Y.J., Gunsalus, K.C. & Szyperski, T. Protein NMR spectroscopy in structural genomics. Nat. Struct. Biol. 7 (Suppl.), 982–985 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Acton, T.B. et al. Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. Methods Enzymol. 394, 210–243 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Alzari, P.M. et al. Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr. D62, 1103–1113 (2006).

    CAS  Google Scholar 

  15. Gileadi, O. The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins. J. Struct. Funct. Genomics 8, 107–119 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gileadi, O. et al. Methods in Molecular Biology. in Structural Proteomics: High-Throughput Methods. Vol. 426 (eds., B. Kobe, M. Guss & T. Huber) 222–246 (Humana Press, Totowa, New Jersey, USA, 2008).

    Google Scholar 

  17. You, J., Cohen, R.E. & Pickart, C.M. Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli. Biotechniques 27, 950–954 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Klock, H.E., Koesema, E.J., Knuth, M.W. & Lesley, S.A. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins published online, doi: 10.1002/prot.21786 (14 November 2007).

  19. Gräslund, S. et al. The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr. Purif. (in the press).

  20. Ginalski, K., Elofsson, A., Fischer, D. & Rychlewski, L. 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 19, 1015–1018 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Ward, J.J., McGuffin, L.J., Bryson, K., Buxton, B.F. & Jones, D.T. The DISOPRED server for the prediction of protein disorder. Bioinformatics 20, 2138–2139 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Yang, Z.R., Thomson, R., McNeil, P. & Esnouf, R.M. RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21, 3369–3376 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Cornvik, T. et al. An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries. Proteins 65, 266–273 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Gao, X. et al. High-throughput limited proteolysis/mass spectrometry for protein domain elucidation. J. Struct. Funct. Genomics 6, 129–134 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Hartley, J.L., Temple, G.F. & Brasch, M.A. DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Aslanidis, C. & de Jong, P.J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Landy, A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu. Rev. Biochem. 58, 913–949 (1989).

    Article  CAS  PubMed  Google Scholar 

  28. Guo, F., Gopaul, D.N. & Van Duyne, G.D. Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse. Proc. Natl. Acad. Sci. USA 96, 7143–7148 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hammarström, M., Hellgren, N., van Den Berg, S., Berglund, H. & Härd, T. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 11, 313–321 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Peti, W. & Page, R. Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr. Purif. 51, 1–10 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Braun, P. & LaBaer, J. High throughput protein production for functional proteomics. Trends Biotechnol. 21, 383–388 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Vincentelli, R. et al. Medium-scale structural genomics: strategies for protein expression and crystallization. Accounts Chem. Res. 36, 165–172 (2003).

    Article  CAS  Google Scholar 

  33. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89 (1990).

    Article  CAS  PubMed  Google Scholar 

  34. Uhlén, M., Forsberg, G., Moks, T., Hartmanis, M. & Nilsson, B. Fusion proteins in biotechnology. Curr. Opin. Biotechnol. 3, 363–369 (1992).

    Article  PubMed  Google Scholar 

  35. Arnau, J., Lauritzen, C., Petersen, G.E. & Pedersen, J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr. Purif. 48, 1–13 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Carrington, J.C. & Dougherty, W.G. A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing. Proc. Natl. Acad. Sci. USA 85, 3391–3395 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Porath, J. Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263–281 (1992).

    Article  CAS  PubMed  Google Scholar 

  38. Nallamsetty, S. & Waugh, D. Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 45, 175–182 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Nallamsetty, S. & Waugh, D.S. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat. Protoc. 2, 383–391 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Waugh, D.S. Making the most of affinity tags. Trends Biotechnol. 23, 316–320 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Carson, M., Johnson, D.H., McDonald, H., Brouillette, C. & Delucas, L.J. His-tag impact on structure. Acta Crystallogr. 63, 295–301 (2007).

    CAS  Google Scholar 

  42. Dubendorff, J.W. & Studier, F.W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59 (1991).

    Article  CAS  PubMed  Google Scholar 

  43. Wycuff, D.R. & Matthews, K.S. Generation of an AraC-araBAD promoter-regulated T7 expression system. Anal. Biochem. 277, 67–73 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Lesley, S.A. High-throughput proteomics: protein expression and purification in the postgenomic world. Protein Expr. Purif. 22, 159–164 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Tunac, J. A new high-aeration capacity shake-flask system. J. Ferm. Bioeng. 68, 15–159 (1989).

    Article  Google Scholar 

  49. Brodsky, O. & Cronin, C.N. Economical parallel protein expression screening and scale-up in Escherichia coli. J. Struct. Funct. Genomics 7, 101–108 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Vera, A., Gonzalez-Montalban, N., Aris, A. & Villaverde, A. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol. Bioeng. 96, 1101–1106 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Berrow, N.S. et al. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr. D62, 1218–1226 (2006).

    CAS  Google Scholar 

  52. Page, R. et al. Scalable high-throughput micro-expression device for recombinant proteins. Biotechniques 37, 364–370 (2004).

    Article  CAS  PubMed  Google Scholar 

  53. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  PubMed  Google Scholar 

  54. Bolanos-Garcia, V.M. & Davies, O.R. Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochim. Biophys. Acta 1760, 1304–1313 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Howell, J.M., Winstone, T.L., Coorssen, J.R. & Turner, R.J. An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins. Proteomics 6, 2050–2069 (2006).

    Article  PubMed  Google Scholar 

  56. Bullock, A.N., Debreczeni, J., Amos, A.L., Knapp, S. & Turk, B.E. Structure and substrate specificity of the Pim-1 kinase. J. Biol. Chem. 280, 41675–41682 (2005).

    Article  CAS  PubMed  Google Scholar 

  57. Alam, M., Ho, S., Vance, D.E. & Lehner, R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase. Protein Expr. Purif. 24, 33–42 (2002).

    Article  CAS  PubMed  Google Scholar 

  58. Kapust, R.B. & Waugh, D.S. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Iakoucheva, L.M., Brown, C.J., Lawson, J.D., Obradovic, Z. & Dunker, A.K. Intrinsic disorder in cell-signaling and cancer-associated proteins. J. Mol. Biol. 323, 573–584 (2002).

    Article  CAS  PubMed  Google Scholar 

  60. Frenkiel-Krispin, D. et al. Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1. J. Biol. Chem. 282, 3458–3464 (2007).

    Article  CAS  PubMed  Google Scholar 

  61. Tolia, N.H. & Joshua-Tor, L. Strategies for protein coexpression in Escherichia coli. Nat. Methods 3, 55–64 (2006).

    Article  CAS  PubMed  Google Scholar 

  62. Romier, C. et al. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr. 62, 1232–1242 (2006).

    Google Scholar 

  63. Bullock, A.N., Debreczeni, J.E., Edwards, A.M., Sundström, M. & Knapp, S. Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc. Natl. Acad. Sci. USA 103, 7637–7642 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Vedadi, M. et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. USA 103, 15835–15840 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Niesen, F.H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221 (2007).

    Article  CAS  PubMed  Google Scholar 

  66. Elleby, B. et al. High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1. Biochim. Biophys. Acta 1700, 199–207 (2004).

    Article  CAS  PubMed  Google Scholar 

  67. Strauss, A. et al. Improved expression of kinases in Baculovirus-infected insect cells upon addition of specific kinase inhibitors to the culture helpful for structural studies. Protein Expr. Purif. 56, 167–176 (2007).

    Article  CAS  PubMed  Google Scholar 

  68. Smith, G.E., Summers, M.D. & Fraser, M.J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3, 2156–2165 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Boettner, M., Prinz, B., Holz, C., Stahl, U. & Lang, C. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J. Biotechnol. 99, 51–62 (2002).

    Article  CAS  PubMed  Google Scholar 

  70. Holz, C., Hesse, O., Bolotina, N., Stahl, U. & Lang, C. A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae. Protein Expr. Purif. 25, 372–378 (2002).

    Article  CAS  PubMed  Google Scholar 

  71. Aricescu, A.R., Lu, W. & Jones, E.Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D62, 1243–1250 (2006).

    CAS  Google Scholar 

  72. Yokoyama, S. Protein expression systems for structural genomics and proteomics. Curr. Opin. Chem. Biol. 7, 39–43 (2003).

    Article  CAS  PubMed  Google Scholar 

  73. Kigawa, T. et al. Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63–68 (2004).

    Article  CAS  PubMed  Google Scholar 

  74. Matsuda, T. et al. Cell-free synthesis of zinc-binding proteins. J. Struct. Funct. Genomics 7, 93–100 (2006).

    Article  CAS  PubMed  Google Scholar 

  75. Endo, Y. & Sawasaki, T. Cell-free expression systems for eukaryotic protein production. Curr. Opin. Biotechnol. 17, 373–380 (2006).

    Article  CAS  PubMed  Google Scholar 

  76. Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S. & Imataka, H. An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr. Purif. 46, 348–357 (2006).

    Article  CAS  PubMed  Google Scholar 

  77. Yokoyama, S., Terwilliger, T.C., Kuramitsu, S., Moras, D. & Sussman, J.L. RIKEN aids international structural genomics efforts. Nature 445, 21 (2007).

    Article  CAS  PubMed  Google Scholar 

  78. Murayama, K. et al. Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism. J. Biol. Chem. 282, 4238–4242 (2007).

    Article  CAS  PubMed  Google Scholar 

  79. Miyazono, K. et al. Novel protein fold discovered in the PabI family of restriction enzymes. Nucleic Acids Res. 35, 1908–1918 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Nishihara, K., Kanemori, M., Yanagi, H. & Yura, T. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66, 884–889 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Wynn, R.M., Song, J.L. & Chuang, D.T. GroEL/GroES promote dissociation/reassociation cycles of a heterodimeric intermediate during alpha(2)beta(2) protein assembly. Iterative annealing at the quaternary structure level. J. Biol. Chem. 275, 2786–2794 (2000).

    Article  CAS  PubMed  Google Scholar 

  82. Kaiser, C.M. et al. Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460 (2006).

    Article  CAS  PubMed  Google Scholar 

  83. Willis, M.S. et al. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Sci. 14, 1818–1826 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Vincentelli, R. et al. High-throughput automated refolding screening of inclusion bodies. Protein Sci. 13, 2782–2792 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Kim, S.H. et al. Structural genomics of minimal organisms and protein fold space. J. Struct. Funct. Genomics 6, 63–70 (2005).

    Article  CAS  PubMed  Google Scholar 

  86. Lesley, S.A. & Wilson, I.A. Protein production and crystallization at the joint center for structural genomics. J. Struct. Funct. Genomics 6, 71–79 (2005).

    Article  CAS  PubMed  Google Scholar 

  87. Kreusch, A. & Lesley, S.A. High throughput cloning, expression and purification technologies. in Genomics, Proteomics, and Vaccines (ed.,G. Grandi) 171–184 (Wiley Press, Chichester, UK, 2004).

    Google Scholar 

  88. McMullan, D. et al. High-throughput protein production for X-ray crystallography and use of size exclusion chromatography to validate or refute computational biological unit predictions. J. Struct. Funct. Genomics 6, 135–141 (2005).

    Article  CAS  PubMed  Google Scholar 

  89. Stols, L., Millard, C.S., Dementieva, I. & Donnelly, M.I. Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination. J. Struct. Funct. Genomics 5, 95–102 (2004).

    Article  CAS  PubMed  Google Scholar 

  90. Geerlof, A. et al. The impact of protein characterization in structural proteomics. Acta Crystallogr. D62, 1125–1136 (2006).

    CAS  Google Scholar 

Download references

Acknowledgements

The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. The New York Structural GenomiX Research Center for Structural Genomics is supported by the US National Institute of General Medical Sciences (U54 GM074945). Work at the MDC was supported by the German Federal Ministry for Education and Research (BMBF) through the Leitprojektverbund Proteinstrukturfabrik and the German National Genome Network (NGFN; FKZ 01GR0471, 01GR0472), and by the Fonds der Chemischen Industrie. The Protein Sample Production Facility is funded by the Helmholtz Association of German Research Centres. The China Structural Genomics Consortium is supported by the National 863 Hi-Tech Research and Development Program of China. The Israel Structural Proteomics Center is supported by The Israel Ministry of Science, Culture and Sport, the Divadol Foundation, the Neuman Foundation, the European Commission Sixth Framework Research and Technological Development Programme 'SPINE2-Complexes' Project under contract 031220. The RIKEN Structural Genomics/Proteomics Initiative was supported by the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan. The Joint Center for Structural Genomics is supported by the US National Institutes of Health (NIH) Protein Structure Initiative grant U54 GM074898 from the NIGMS. The Northeast Structural Genomics Consortium is supported by the NIH NIGMS (U54-GM074958). The Midwest Center for Structural Genomics is supported by the NIH (GM074942) and by the US Department of Energy, Office of Biological and Environmental Research (DE-AC02-06CH11357). The Oxford Protein Production Facility is funded by the UK Medical Research Council and Biotechnology and Biological Sciences Research Council. SPINE2-Complexes is funded by the European Commission (contract 031220) under the Framework 6 RTD Programme and is coordinated from the Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Oxford, UK. The Berkeley Structural Genomics Center is supported by the NIH (GM62412). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or the NIH.

Author information

Author notes

  1. B Martin Hallberg, Konrad Büssow & Kristin C Gunsalus

    Present address: Present addresses: Structural Biology, Helmoltz Center for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany (K.B.), and Department of Biology, and Center for Genomics and Systems Biology, New York University, 1009 Sliver Center, 100 Washington Square East, New York, NY 10003, USA (K.C.G.).,

  2. A complete list of authors appears at the end of this paper.

Authors and Affiliations

  1. Karolinska Institutet, Schéeles väg 2, 171 77, Stockholm, Sweden

    Susanne Gräslund, Pär Nordlund, Johan Weigelt & B Martin Hallberg

  2. University of Oxford, Old Road Campus, Roosevelt Drive, Headington, OX3 7DQ, Oxford, UK

    James Bray, Opher Gileadi, Stefan Knapp & Udo Oppermann

  3. University of Toronto, 100 College St., Toronto, M5G 1L6, Ontario, Canada

    Cheryl Arrowsmith, Raymond Hui, Jinrong Ming, Sirano dhe-Paganon, Hee-won Park, Alexei Savchenko, Adelinda Yee & Aled Edwards

  4. Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Case 932, 163 Avenue de Luminy, Marseille, 13288, Cedex 09, France

    Renaud Vincentelli & Christian Cambillau

  5. Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, 351A Donner Laboratory, Berkeley, 94720, California, USA

    Rosalind Kim & Sung-Hou Kim

  6. Tsinghua University, 100084, Beijing, China

    Zihe Rao

  7. University of Science and Technology of China, 230027, Hefei, China

    Yunyu Shi

  8. Los Alamos National Laboratory, Mailstop M888, Los Alamos, 87507, New Mexico, USA

    Thomas C Terwilliger, Chang-Yub Kim, Li-Wei Hung & Geoffrey S Waldo

  9. Weizmann Institute of Science, 2 Herzel Street, 76100, Rehovot, Israel

    Yoav Peleg, Shira Albeck, Tamar Unger, Orly Dym, Jaime Prilusky & Joel L Sussman

  10. The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, 92037, California, USA

    Ray C Stevens, Scott A Lesley & Ian A Wilson

  11. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121, California, USA

    Scott A Lesley & Ian A Wilson

  12. Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, 60439, Illinois, USA

    Andrzej Joachimiak, Frank Collart, Irina Dementieva, Mark I Donnelly, William H Eschenfeldt, Youngchang Kim, Lucy Stols, Ruying Wu & Min Zhou

  13. SGX Pharmaceuticals, Inc., 10505 Roselle Street, San Diego, 92121, California, USA

    Stephen K Burley, J Spencer Emtage, J Michael Sauder, Devon Thompson, Kevin Bain, John Luz, Tarun Gheyi, Fred Zhang & Shane Atwell

  14. Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Steven C Almo, Jeffrey B Bonanno & Andras Fiser

  15. Biology Department, 463, Brookhaven National Laboratory, Upton, 11973, New York, USA

    Sivasubramanian Swaminathan & F William Studier

  16. Case Western Reserve University, 10900 Euclid Ave., Cleveland, 44016, Ohio, USA

    Mark R Chance

  17. Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, San Francisco, 94143, California, USA

    Andrej Sali

  18. Center for Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane, Piscataway, 08854, New Jersey, USA

    Thomas B Acton, Rong Xiao, Li Zhao, Li Chung Ma, Kellie Cunningham, Masayori Inouye, Stephen Anderson, Heleema Janjua, Ritu Shastry, Chi Kent Ho, Dongyan Wang, Huang Wang, Mei Jiang, Gaetano T Montelione & Kristin C Gunsalus

  19. Department of Biological Sciences, Columbia University, 701 Fairchild Building, MC 2451, New York, 10027, New York, USA

    John F Hunt & Liang Tong

  20. Wellcome Trust Centre for Human Genetics, Roosevelt Drive, OX37BN, Oxford, UK

    David I Stuart, Raymond J Owens & Susan Daenke

  21. Max Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13092, Berlin, Germany

    Anja Schütz, Udo Heinemann & Konrad Büssow

  22. Protein Research Group, Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, 230-0045, Yokohama, Japan

    Shigeyuki Yokoyama

  23. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, OX37BN, Oxford, UK

    David I Stuart, Raymond J Owens & Susan Daenke

Consortia

Structural Genomics Consortium

  • Susanne Gräslund
  • , Pär Nordlund
  • , Johan Weigelt
  • , B Martin Hallberg
  • , James Bray
  • , Opher Gileadi
  • , Stefan Knapp
  • , Udo Oppermann
  • , Cheryl Arrowsmith
  • , Raymond Hui
  • , Jinrong Ming
  • , Sirano dhe-Paganon
  • , Hee-won Park
  • , Alexei Savchenko
  • , Adelinda Yee
  •  & Aled Edwards

Architecture et Fonction des Macromolécules Biologiques

  • Renaud Vincentelli
  •  & Christian Cambillau

Berkeley Structural Genomics Center

  • Rosalind Kim
  •  & Sung-Hou Kim

China Structural Genomics Consortium

  • Zihe Rao
  •  & Yunyu Shi

Integrated Center for Structure and Function Innovation

  • Thomas C Terwilliger
  • , Chang-Yub Kim
  • , Li-Wei Hung
  •  & Geoffrey S Waldo

Israel Structural Proteomics Center

  • Yoav Peleg
  • , Shira Albeck
  • , Tamar Unger
  • , Orly Dym
  • , Jaime Prilusky
  •  & Joel L Sussman

Joint Center for Structural Genomics

  • Ray C Stevens
  • , Scott A Lesley
  •  & Ian A Wilson

Midwest Center for Structural Genomics

  • Andrzej Joachimiak
  • , Frank Collart
  • , Irina Dementieva
  • , Mark I Donnelly
  • , William H Eschenfeldt
  • , Youngchang Kim
  • , Lucy Stols
  • , Ruying Wu
  •  & Min Zhou

New York Structural GenomiX Research Center for Structural Genomics

  • Stephen K Burley
  • , J Spencer Emtage
  • , J Michael Sauder
  • , Devon Thompson
  • , Kevin Bain
  • , John Luz
  • , Tarun Gheyi
  • , Fred Zhang
  • , Shane Atwell
  • , Steven C Almo
  • , Jeffrey B Bonanno
  • , Andras Fiser
  • , Sivasubramanian Swaminathan
  • , F William Studier
  • , Mark R Chance
  •  & Andrej Sali

Northeast Structural Genomics Consortium

  • Thomas B Acton
  • , Rong Xiao
  • , Li Zhao
  • , Li Chung Ma
  • , John F Hunt
  • , Liang Tong
  • , Kellie Cunningham
  • , Masayori Inouye
  • , Stephen Anderson
  • , Heleema Janjua
  • , Ritu Shastry
  • , Chi Kent Ho
  • , Dongyan Wang
  • , Huang Wang
  • , Mei Jiang
  •  & Gaetano T Montelione

Oxford Protein Production Facility

  • David I Stuart
  • , Raymond J Owens
  •  & Susan Daenke

Protein Sample Production Facility, Max Delbrück Center for Molecular Medicine

  • Anja Schütz
  •  & Udo Heinemann

RIKEN Structural Genomics/Proteomics Initiative

  • Shigeyuki Yokoyama

SPINE2-Complexes

  • Konrad Büssow
  •  & Kristin C Gunsalus

Corresponding author

Correspondence to Aled Edwards.

Supplementary information

Supplementary Methods, Table and Figures

Supplementary Methods, Supplementary Table 1, Supplementary Figures 1–2 (PDF 539 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Structural Genomics Consortium., Architecture et Fonction des Macromolécules Biologiques., Berkeley Structural Genomics Center. et al. Protein production and purification. Nat Methods 5, 135–146 (2008). https://doi.org/10.1038/nmeth.f.202

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.f.202

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing