Purification of Nitrogenase Proteins

  • Chi-Chung Lee
  • Markus W. RibbeEmail author
  • Yilin HuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1876)


A major hurdle in the studies of nitrogenase, one of the most complicated metalloenzymes known to date, is to obtain large amounts of intact, active proteins. Nitrogenase and related proteins are often multimeric and consist of metal centers that are critical for their activities. Most notably, the well-studied MoFe protein of Mo-nitrogenase is a heterotetramer that houses two of the most complicated metal clusters found in nature, the P-cluster and the FeMoco (or M-cluster). The structural complexity of these proteins and the oxygen sensitivity of their associated metal clusters, along with the demand for large amounts of high-quality proteins in most downstream analyses, make large-scale, high-yield purification of fully competent nitrogenase proteins a formidable task and yet, at the same time, a prerequisite for the success of nitrogenase research. This chapter highlights several methods that have been developed over the past few decades chiefly for the purification of naturally expressed nitrogenase in the diazotroph Azotobacter vinelandii. In addition, purification and Fe-S reconstitution strategies are also outlined for the heterologously expressed nitrogenase proteins in Escherichia coli.

Key words

Anaerobic protein purification Nitrogenase MoFe protein Fe protein VFe protein Weak anion-exchange chromatography (WAEC) Gel filtration Size-exclusion chromatography (SEC) Immobilized metal-affinity chromatography (IMAC) Heterologous expression Fe–S reconstitution 



The authors are supported by the US Department of Energy (Basic Energy Sciences) grant DE-SC0016510 (to Y.H. and M.W.R.).


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiochemistryUniversity of California, IrvineIrvineUSA
  2. 2.Department of ChemistryUniversity of California, IrvineIrvineUSA

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