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Purification of Nitrogenase Proteins

  • Jared A. Wiig
  • Chi-Chung Lee
  • Aaron W. Fay
  • Yilin Hu
  • Markus W. RibbeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 766)

Abstract

Nitrogenase is one of the most complex enzymes known to date. The extensively studied molybdenum nitrogenase consists of two protein components and three metal centers that are critical for nitrogenase activity. The inherent complexity of this enzyme system, which is further compounded by the sensitivity of the metal clusters toward oxygen, makes the large-scale purification of fully active nitrogenase proteins a formidable task. This chapter highlights several methods that have been developed for the purification of nitrogenase proteins over the past few decades. Techniques used include weak anion exchange chromatography, size exclusion chromatography, and immobilized metal affinity chromatography. These methods can be selectively applied to nitrogenase variants and other related proteins.

Key words

Anaerobic protein purification nitrogenase MoFe protein Fe protein VFe protein weak anion exchange chromatography (WAEC) gel filtration immobilized metal affinity chromatography (IMAC) 

Notes

Acknowledgments

The authors are supported by National Institutes of Health grant GM 67626 (M.W.R.) and Herman Frasch Foundation grant 617-HF07 (M.W.R.).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jared A. Wiig
    • 1
  • Chi-Chung Lee
    • 1
  • Aaron W. Fay
    • 1
  • Yilin Hu
    • 1
  • Markus W. Ribbe
    • 1
    Email author
  1. 1.Department of Molecular Biology and BiochemistryUniversity of CaliforniaIrvineUSA

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