Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system

  • Nathan P. Cowieson
  • Pawel Listwan
  • Mareike Kurz
  • Anna Aagaard
  • Timothy Ravasi
  • Christine Wells
  • Thomas Huber
  • David A. Hume
  • Bostjan Kobe
  • Jennifer L. Martin
Article

Abstract

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.

Keywords

bacterial expression mammalian proteins medium-throughput protein expression screening recombinatorial cloning 

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References

  1. 1.
    Stevens, R.C., Wilson, I.A. 2001Science293519520CrossRefPubMedGoogle Scholar
  2. 2.
    Lesley, S.A., Kuhn, P., Godzik, A., Deacon, A.M., Mathews, I., Kreusch, A.,  et al. 2002Proc. Natl. Acad. Sci. USA991166411669CrossRefPubMedGoogle Scholar
  3. 3.
    Canaves, J.M., Page, R., Wilson, I.A., Stevens, R.C. 2004J. Mol. Biol.344977991CrossRefPubMedGoogle Scholar
  4. 4.
    Yokoyama, S., Hirota, H., Kigawa, T., Yabuki, T., Shirouzu, M., Terada, T.,  et al. 2000Nat. Struct. Biol.7943945PubMedGoogle Scholar
  5. 5.
    Vincentelli, R., Bignon, C., Gruez, A., Canaan, S., Sulzenbacher, G., Tegoni, M., Campanacci, V., Cambillau, C. 2003Acc. Chem. Res.36165172CrossRefPubMedGoogle Scholar
  6. 6.
    Braun, P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E., LaBaer, J. 2002Proc. Natl. Acad. Sci. USA9926542659CrossRefPubMedGoogle Scholar
  7. 7.
    Chen, L., Oughtred, R., Berman, H.M., Westbrook, J. 2004Bioinformatics2028602862CrossRefPubMedGoogle Scholar
  8. 8.
    Yokoyama, S. 2003Curr. Opin. Chem. Biol.73943CrossRefPubMedGoogle Scholar
  9. 9.
    Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H., Edwards, A.M. 2000Prog. Biophys. Mol. Biol.73339345CrossRefPubMedGoogle Scholar
  10. 10.
    Huang, R.Y., Boulton, S.J., Vidal, M., Almo, S.C., Bresnick, A.R., Chance, M.R. 2003Biochem. Biophys. Res. Commun.307928934CrossRefPubMedGoogle Scholar
  11. 11.
    Heinemann, U., Bussow, K., Mueller, U., Umbach, P. 2003Acc. Chem. Res.36157163CrossRefPubMedGoogle Scholar
  12. 12.
    Widmann, M., Christen, P. 2000J. Biol. Chem.2751861918622CrossRefPubMedGoogle Scholar
  13. 13.
    Tresaugues, L., Collinet, B., Minard, P., Henckes, G., Aufrere, R., Blondeau, K.,  et al. 2004J. Struct. Funct. Genomics5195204CrossRefPubMedGoogle Scholar
  14. 14.
    You, J., Cohen, R.E., Pickart, C.M. 1999Biotechniques27950954PubMedGoogle Scholar
  15. 15.
    Endo, Y., Sawasaki, T. 2004J. Struct. Funct. Genomics54557CrossRefPubMedGoogle Scholar
  16. 16.
    Holz, C., Hesse, O., Bolotina, N., Stahl, U., Lang, C. 2002Protein Expr Purif.25372378CrossRefPubMedGoogle Scholar
  17. 17.
    Albala, J.S., Franke, K., McConnell, I.R., Pak, K.L., Folta, P.A., Rubinfeld, B., Davies, A.H., Lennon, G.G., Clark, R. 2000J. Cell. Biochem.80187191CrossRefPubMedGoogle Scholar
  18. 18.
    Wells, C.A., Ravasi, T., Sultana, R., Yagi, K., Carninci, P., Bono, H., Faulkner, G., Okazaki, Y., Quackenbush, J., Hume, D.A., Lyons, P.A. 2003Genome Res1313601365CrossRefPubMedGoogle Scholar
  19. 19.
    Wells, C.A., Ravasi, T., Faulkner, G.J., Carninci, P., Okazaki, Y., Hayashizaki, Y., Sweet, M., Wainwright, B.J., Hume, D.A. 2003BMC Immunol45CrossRefPubMedGoogle Scholar
  20. 20.
    Okazaki, Y., Furuno, M., Kasukawa, T., Adachi, J., Bono, H., Kondo, S.,  et al. 2002Nature420563573CrossRefPubMedGoogle Scholar
  21. 21.
    Chen, G.Q., Gouaux, E. 1997Proc. Natl.Acad.Sci. USA941343113436CrossRefPubMedGoogle Scholar
  22. 22.
    Moy, S., Dieckman, L, Schiffer, M, Maltsev, N, Yu, G.X., Collart, F.R. 2004J. Struct. Funct.Genomics5103109Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Nathan P. Cowieson
    • 1
  • Pawel Listwan
    • 2
    • 3
    • 5
  • Mareike Kurz
    • 1
  • Anna Aagaard
    • 1
  • Timothy Ravasi
    • 1
    • 3
    • 5
  • Christine Wells
    • 1
    • 3
    • 5
  • Thomas Huber
    • 4
  • David A. Hume
    • 1
    • 3
    • 5
  • Bostjan Kobe
    • 1
    • 2
    • 5
  • Jennifer L. Martin
    • 1
    • 2
    • 5
  1. 1.Institute for Molecular BioscienceAustralia
  2. 2.School of Molecular and Microbial SciencesAustralia
  3. 3.Cooperative Research Centre for Chronic Inflammatory DiseasesAustralia
  4. 4.Department of MathematicsAustralia
  5. 5.ARC Special Research Centre for Functional and Applied Genomics, Queensland Bioscience PrecinctUniversity of QueenslandBrisbaneAustralia

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