Advertisement

High Throughput Production of Recombinant Human Proteins for Crystallography

  • Opher Gileadi
  • Nicola A. Burgess-Brown
  • Steve M. Colebrook
  • Georgina Berridge
  • Pavel Savitsky
  • Carol E. A. Smee
  • Peter Loppnau
  • Catrine Johansson
  • Eidarus Salah
  • Nadia H. Pantic
Part of the Methods in Molecular Biology™ book series (MIMB, volume 426)

This chapter presents in detail the process used in high throughput bacterial production of recombinant human proteins for crystal structure determination. The core principles are: (1) Generating at least 10 truncated constructs from each target gene. (2) Ligation-independent cloning (LIC) into a bacterial expression vector. All proteins are expressed with an N-terminal, TEV protease cleavable fusion peptide. (3) Small-scale test expression to identify constructs producing soluble protein. (4) Liter-scale production in shaker flasks. (5) Purification by Ni-affinity chromatography and gel filtration. (6) Protein characterization and preparation for crystallography. The chapter also briefly presents alternative procedures, to be applied based on specific knowledge of protein families or when the core protocol is unsatisfactory. This scheme has been applied to more than 550 human proteins (>10,000 constructs) and has resulted in the deposition of 112 unique structures. The methods presented do not depend on specialized equipment or robotics; hence, they provide an effective approach for handling individual proteins in a regular research lab.

Keywords

Column Volume Porous Film Immobilize Metal Affinity Chromatography Glycerol Stock Test Expression 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Makrides, S. C. (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538.PubMedGoogle Scholar
  2. 2.
    Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C. H., and Edwards, A. M. (2000) Structural proteomics: prospects for high throughput sample preparation. Prog. Biophys. Mol. Biol. 73, 339–345.CrossRefPubMedGoogle Scholar
  3. 3.
    Edwards, A. M., Arrowsmith, C. H., Christendat, D., Dharamsi, A., Friesen, J. D., Greenblatt, J. F., and Vedadi, M. (2000) Protein production: feeding the crystallog-raphers and NMR spectroscopists. Nat. Struct. Biol. 7, 970–972.CrossRefPubMedGoogle Scholar
  4. 4.
    Gerstein, M., Edwards, A., Arrowsmith, C. H., and Montelione, G. T. (2003) Structural genomics: current progress. Science 299, 1663.CrossRefPubMedGoogle Scholar
  5. 5.
    Stevens, R. C. (2000) Design of high throughput methods of protein production for structural biology. Structure 8, R177–185.CrossRefPubMedGoogle Scholar
  6. 6.
    Stevens, R. C., Yokoyama, S., and Wilson, I. A. (2001) Global efforts in structural genomics. Science 294, 89–92.CrossRefPubMedGoogle Scholar
  7. 7.
    Vincentelli, R., Bignon, C., Gruez, A., Canaan, S., Sulzenbacher, G., Tegoni, M., Campanacci, V., and Cambillau, C. (2003) Medium-scale structural genomics: strategies for protein expression and crystallization. Acc. Chem. Res. 36, 165–172.CrossRefPubMedGoogle Scholar
  8. 8.
    Yakunin, A. F., Yee, A. A., Savchenko, A., Edwards, A. M., and Arrowsmith, C. H. (2004) Structural proteomics: a tool for genome annotation. Curr. Opin. Chem. Biol. 8, 42–48.CrossRefPubMedGoogle Scholar
  9. 9.
    Yee, A., Pardee, K., Christendat, D., Savchenko, A., Edwards, A. M., and Arrowsmith, C. H. (2003) Structural proteomics: toward high throughput structural biology as a tool in functional genomics. Acc. Chem. Res. 36, 183–189.CrossRefPubMedGoogle Scholar
  10. 10.
    Albeck, S., Burstein, Y., Dym, O., Jacobovitch, Y., Levi, N., Meged, R., Michael, Y., Peleg, Y., Prilusky, J., Schreiber, G., Silman, I., Unger, T., and Sussman, J. L. (2005) Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC). This paper was presented at ICCBM10. Acta Crystallogr. D Biol. Crystallogr. 61, 1364–1372.CrossRefPubMedGoogle Scholar
  11. 11.
    Mehlin, C., Boni, E., Buckner, F. S., Engel, L., Feist, T., Gelb, M. H., Haji, L., Kim, D., Liu, C., Mueller, N., Myler, P. J., Reddy, J. T., Sampson, J. N., Subramanian, E., Van Voorhis, W. C., Worthey, E., Zucker, F., and Hol, W. G. (2006) Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes. Mol. Biochem. Parasitol. 148, 144–160.CrossRefPubMedGoogle Scholar
  12. 12.
    Dale, G. E., Oefner, C., and D'Arcy, A. (2003) The protein as a variable in protein crystallization. J. Struct. Biol. 142, 88–97.CrossRefGoogle Scholar
  13. 13.
    Baneyx, F. (1999) Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421.CrossRefPubMedGoogle Scholar
  14. 14.
    Cornvik, T., Dahlroth, S. L., Magnusdottir, A., Flodin, S., Engvall, B., Lieu, V., Ekberg, M., and Nordlund, P. (2006) An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries. Proteins 65, 266–273.CrossRefPubMedGoogle Scholar
  15. 15.
    Cornvik, T., Dahlroth, S. L., Magnusdottir, A., Herman, M. D., Knaust, R., Ekberg, M., and Nordlund, P. (2005) Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli. Nat. Methods 2, 507–509.CrossRefPubMedGoogle Scholar
  16. 16.
    Hanahan, D., Jessee, J., and Bloom, F. R. (1991) Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 204, 63–113.CrossRefPubMedGoogle Scholar
  17. 17.
    Stols, L., Gu, M., Dieckman, L., Raffen, R., Collart, F. R., and Donnelly, M. I. (2002) A new vector for high throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein. Expr. Purif. 25, 8–15.CrossRefPubMedGoogle Scholar
  18. 18.
    Page, R., Moy, K., Sims, E. C., Velasquez, J., McManus, B., Grittini, C., Clayton, T. L., and Stevens, R. C. (2004) Scalable high throughput micro-expression device for recombinant proteins. Biotechniques 37, 364–368.PubMedGoogle Scholar
  19. 19.
    Millard, C. S., Stols, L., Quartey, P., Kim, Y., Dementieva, I., and Donnelly, M. I. (2003) A less laborious approach to the high throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles. Protein. Expr. Purif. 29, 311–320.PubMedGoogle Scholar
  20. 20.
    Burgess, R. (1991) Use of polyethylenimine in purification of DNA-binding proteins. Methods Enzymol 208, 3–10.CrossRefPubMedGoogle Scholar
  21. 21.
    Feaver, W. J., Gileadi, O., and Kornberg, R. D. (1991) Purification and characterization of yeast RNA polymerase II transcription factor b. J. Biol. Chem. 266, 19000–19005.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Opher Gileadi
    • 1
  • Nicola A. Burgess-Brown
    • 1
  • Steve M. Colebrook
    • 1
  • Georgina Berridge
    • 1
  • Pavel Savitsky
    • 1
  • Carol E. A. Smee
    • 1
  • Peter Loppnau
    • 2
  • Catrine Johansson
    • 1
  • Eidarus Salah
    • 1
  • Nadia H. Pantic
    • 1
  1. 1.The Structural Genomics Consortium, Botnar Research CentreUniversity of OxfordOxfordUK
  2. 2.The Structural Genomics ConsortiumUniversity of TorontoTorontoCanada

Personalised recommendations