Nitrogenases are complex two-component metalloenzymes that catalyze biological nitrogen fixation. Three different nitrogenase types are found in the model nitrogen-fixing microbe Azotobacter vinelandii. In the case of the Mo-dependent enzyme, the two catalytic partners are referred to as the Fe protein and MoFe protein. In addition to genes encoding the catalytic components, there are a total of 68 other gene products known to be variously involved in producing, activating, protecting, sustaining, and regulating formation of the Mo-dependent nitrogenase. In order to support experiments designed to gain insight into the catalytic mechanism and assembly of nitrogenase, four different affinity-based purification protocols have been developed. These include an improved Co2 +-based Immobilized Metal Affinity Chromatography (IMAC) method for the purification of MoFe protein, a newly developed StrepTactin Affinity Chromatography (STAC) method for the purification of MoFe protein and its assembly intermediates, a combined IMAC and STAC method for isolation of highly pure MoFe protein, and a STAC-based bait–prey method for isolation of complexes variously involved in the maturation process.
Article
Application of Affinity Purification Methods for Analysis of the Nitrogenase System from Azotobacter Vinelandii
Methods in Enzymology
Document Type
Article
Publication Date
11-23-2018
Publisher
Academic Press
Abstract
Citation Information
Jiménez-Vicente, E., Martin Del Campo, J. S., Yang, Z.-Y., Cash, V. L., Dean, D. R., and Seefeldt, L. C. (2018) Chapter Nine -Application of affinity purification methods for analysis of the nitrogenase system from Azotobacter vinelandii, in Methods in Enzymology (Armstrong, F., Ed.), pp 231–255. Academic Press.