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High-throughput Protein Production for X-ray Crystallography and Use of Size Exclusion Chromatography to Validate or Refute Computational Biological Unit Predictions

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Journal of Structural and Functional Genomics

Abstract

The production of large numbers of highly purified proteins for X-ray crystallography is a significant bottleneck in structural genomics. At the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org), specific automated protein expression, purification, and analytical methods are being utilized to study the proteome of Thermotoga maritima. Anion exchange and size exclusion chromatography (SEC), intended for the production of highly purified proteins, have been automated and the procedures are described here in detail. Analytical SEC has been included as a standard quality control test. A biological unit (BU) is the macromolecule that has been proven or is presumed to be functional. Correct assignment of BUs from protein structures can be difficult. BU predictions obtained via the Protein Quaternary Structure file server (PQS; http://pqs.ebi.ac.uk/) were compared to SEC data for 16 representative T. maritima proteins whose structures were solved at the JCSG, revealing an inconsistency in five cases. Herein, we report that SEC can be used to validate or disprove PQS-derived oligomeric models. A substantial amount of associated SEC and structural data should enable us to use certain PQS parameters to gauge the accuracy of these computational models and to generally improve their predictions.

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Authors and Affiliations

  1. Joint Center for Structural Genomics, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA, 92121-1127, USA

    Daniel McMullan, Kevin Quijano, Polat Abdubek, Edward Nigoghossian, Justin Haugen, Heath E. Klock, Juli Vincent, Joanna Hale, Jessica Paulsen & Scott A. Lesley

  2. Joint Center for Structural Genomics, Center for Research in Biological Systems, University of California, San Diego, La Jolla, 9500 Gilman Drive, CA, 92093–0527, USA

    Jaume M. Canaves

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  1. Daniel McMullan
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  2. Jaume M. Canaves
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  3. Kevin Quijano
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  4. Polat Abdubek
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  5. Edward Nigoghossian
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  6. Justin Haugen
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  7. Heath E. Klock
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  8. Juli Vincent
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  9. Joanna Hale
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  10. Jessica Paulsen
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  11. Scott A. Lesley
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Correspondence to Scott A. Lesley.

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McMullan, D., Canaves, J.M., Quijano, K. 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). https://doi.org/10.1007/s10969-005-2898-1

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  • DOI: https://doi.org/10.1007/s10969-005-2898-1

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