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Published on: October 2, 2012
Characterization of the Azotobacter vinelandii nitrogenase complex expressed in Escherichia coli toward further
Yusuke Ito1,2, Daisuke Yoshidome1, Makoto Hidaka1
1Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo.
Abstract:
We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD2K2, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD2K2 component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD2K2 formation, effectively increased nitrogenase activity.
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