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Journal of Structural and Functional Genomics

, Volume 6, Issue 2–3, pp 143–147 | Cite as

High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

  • Won Bae Jeon
  • David J. Aceti
  • Craig A. Bingman
  • Frank C. Vojtik
  • Andrew C. Olson
  • Jason M. Ellefson
  • Janet E. McCombs
  • Hassan K. Sreenath
  • Paul G. Blommel
  • Kory D. Seder
  • Brendan T. Burns
  • Holalkere V. Geetha
  • Amy C. Harms
  • Grzegorz Sabat
  • Michael R. Sussman
  • Brian G. Fox
  • George N. PhillipsJr.
Article

Abstract

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)6-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated ÄKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)6-MBP tags by TEV protease, (His)6-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and 15N and 13C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

Key words

Arabidopsis proteins His-tag nickel affinity purification high-throughput structural genomics 

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References

  1. 1.
    Kapust, R.B., Waugh, D.S. 1999Protein Sci.816681674PubMedGoogle Scholar
  2. 2.
    Amersham Biosciences (2004) Biodirectory, 558–568Google Scholar
  3. 3.
    New England Biolabs, 2005–2006 Catalog & Technical Reference, p 175Google Scholar
  4. 4.
    Blommel, P.G., Fox, B.G. 2005Anal. Biochem3367586CrossRefPubMedGoogle Scholar
  5. 5.
    Thao, S, Zhao, Q., Kimball,  T., Steffan, E., Blommel, P.G., Riters,  M., Newman, C.S., Fox, B.G., Wrobel, R.L. 2004J. Struct. Funct. Genom5267276CrossRefGoogle Scholar
  6. 6.
    Sreenath, H.K., Bingman, C.A., Buchan, B.W., Seder, K.D., Burns, B.T., Geetha, H.V., Jeon, W.B., Vojtik, F.C, Aceti, D.J., Frederick, R.O., Phillips, G.N.,Jr., Fox, B.G. 2005Protein Expression Purif40256267CrossRefGoogle Scholar
  7. 7.
    Phan, J., Zdanov, A., Evdokimov, A.G., Tropea, J.E., Peters, H.P.K., Kapust, R.B., Li, M., Wlodawer, A., Waugh, D.S. 2002J. Biol. Chem.2775056450572CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Won Bae Jeon
    • 1
  • David J. Aceti
    • 1
  • Craig A. Bingman
    • 1
  • Frank C. Vojtik
    • 1
  • Andrew C. Olson
    • 1
  • Jason M. Ellefson
    • 1
  • Janet E. McCombs
    • 1
  • Hassan K. Sreenath
    • 1
  • Paul G. Blommel
    • 1
  • Kory D. Seder
    • 1
  • Brendan T. Burns
    • 1
  • Holalkere V. Geetha
    • 1
  • Amy C. Harms
    • 1
  • Grzegorz Sabat
    • 1
  • Michael R. Sussman
    • 1
  • Brian G. Fox
    • 1
  • George N. PhillipsJr.
    • 1
  1. 1.The Center for Eukaryotic Structural GenomicsUniversity of Wisconsin-MadisonMadisonUSA

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