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

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Journal of Structural and Functional Genomics

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.

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Authors and Affiliations

  1. The Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Bobcock Drive, Madison, Wisconsin, 53706-1549, USA

    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. Phillips Jr.

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  1. Won Bae Jeon
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  2. David J. Aceti
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  3. Craig A. Bingman
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  4. Frank C. Vojtik
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  5. Andrew C. Olson
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  6. Jason M. Ellefson
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  7. Janet E. McCombs
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  8. Hassan K. Sreenath
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  9. Paul G. Blommel
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  10. Kory D. Seder
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  11. Brendan T. Burns
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  12. Holalkere V. Geetha
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  13. Amy C. Harms
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  14. Grzegorz Sabat
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  15. Michael R. Sussman
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  16. Brian G. Fox
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  17. George N. Phillips Jr.
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Corresponding author

Correspondence to George N. Phillips Jr..

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Jeon, W.B., Aceti, D.J., Bingman, C.A. et al. High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics. J Struct Funct Genomics 6, 143–147 (2005). https://doi.org/10.1007/s10969-005-1908-7

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  • DOI: https://doi.org/10.1007/s10969-005-1908-7

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