Abstract 2587 Recombinant Expression and Purification of A. vinelandii CowN in E. coli

Authors

Julie Takei, Chapman University
Cedric P. Owens, Chapman UniversityFollow
Katie Sanders, Chapman University
Neeraja Gajendran, Chapman University

Document Type

Article

Publication Date

6-13-2025

Abstract

The goal of this project is to study the protein CowN in the diazotroph Azotobacter vinelandii (Av) in comparison to CowN in Gluconacetobacter diazotrophicus (Gd). Nitrogenase is an enzyme that catalyzes the reduction of nitrogen gas into ammonia in diazotrophic bacteria. In many diazotrophs, the protein CowN prevents nitrogenase inhibition from the environmental gas carbon monoxide (CO). Previous work studied CowN in the diazotroph G. diazotrophicus. In G. diazotrophicus, CowN binds to nitrogenase and weakens CO binding to its active site. The mechanism of CowN has only been investigated in G. diazotrophicus. Thus, we are interested in determining if CowN's mechanism is similar in other diazotrophs and how the structure of CowN differs between them. This poster describes our efforts to express and purify Av-CowN. Av-CowN was cloned into a pET28a expression vector and expressed recombinantly in E. coli. The protein was then purified in a two-step process. While small amounts of Av-CowN were obtained, it did not appear active since it did not protect Av-nitrogenase from CO. Furthermore, Av-CowN was not folded, suggesting that it is either expressed as a misfolded protein or was damaged during purification. Future work will focus on expressing the protein using cell-free expression systems.

Comments

Originally published in Journal of Biological Chemistry, volume 301, issue 5, supplement, in 2025. https://doi.org/10.1016/j.jbc.2025.110011

Recommended Citation

Takei, J., Owens, C., Sanders, K., Gajendran, N., 2025. Abstract 2587 Recombinant expression and purification of A. vinelandii CowN in E. coli. Journal of Biological Chemistry 301. https://doi.org/10.1016/j.jbc.2025.110011

Copyright

The American Society for Biochemistry and Molecular Biology, Inc.

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.