Application of Affinity Purification Methods for Analysis of the Nitrogenase System from Azotobacter Vinelandii
Document Type
Article
Journal/Book Title
Methods in Enzymology
Publication Date
11-23-2018
Publisher
Academic Press
Volume
613
First Page
231
Last Page
255
Abstract
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.
Recommended Citation
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.
