Automation of protein purification for structural genomics

  • Youngchang Kim
  • Irina Dementieva
  • Min Zhou
  • Ruiying Wu
  • Lour Lezondra
  • Pearl Quartey
  • Grazyna Joachimiak
  • Olga Korolev
  • Hui Li
  • Andrzej Joachimiak
Article

Abstract

A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. ‘Structural-biology-grade’ proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.

abbreviations

MCSG — Midwest Center for Structural Genomics; IMAC — immobilized metal affinity chromatography; TEV — tobacco etch virus; —β-ME —β-mercaptoethanol; DTT — dithiothreitol; EDTA — ethylenediaminetetraacetate; SDS-PAGE — polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

affinity chromatography automation protein purification structural genomics 

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Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Youngchang Kim
    • 1
  • Irina Dementieva
    • 1
  • Min Zhou
    • 1
  • Ruiying Wu
    • 1
  • Lour Lezondra
    • 1
  • Pearl Quartey
    • 1
  • Grazyna Joachimiak
    • 1
  • Olga Korolev
    • 1
  • Hui Li
    • 1
  • Andrzej Joachimiak
    • 1
  1. 1.Biosciences Division and Structural Biology Center, Argonne National LaboratoryArgonneUSA

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