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Protein Expr Purif. 2019 Mar 13;160:11-18. doi: 10.1016/j.pep.2019.03.005. [Epub ahead of print]

The purification of the σFpvI/FpvR20 and σPvdS/FpvR20 protein complexes is facilitated at room temperature.

Author information

1
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
2
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
3
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia. Electronic address: M.Maher@latrobe.edu.au.

Abstract

Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σFpvI and σPvdS bind specifically to the antisigma factor FpvR. These proteins are an essential component of the pyoverdine-based system for iron uptake in Pseudomonas aeruginosa. Due to the uniqueness of this system, where the activities of both the σFpvI and σPvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR20 and the σFpvI and σPvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σFpvI and σPvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His6FpvI/FpvR1-67, His6FpvI/FpvR1-89, His6PvdS/FpvR1-67 and His6PvdS/FpvR1-89 protein complexes (where FpvR1-67 and FpvR1-89 are truncated versions of FpvR20) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His6FpvI/FpvR1-67 and His6PvdS/FpvR1-67 complexes. A comparison of these values with those previously reported for the His6FpvI/FpvR1-89 and His6PvdS/FpvR1-89 complexes is made.

KEYWORDS:

Analytical ultracentrifugation (AUC); Circular dichroism (CD) spectroscopy; Iron uptake; Pseudomonas aeruginosa; Pyoverdine; Sigma/antisigma factors

PMID:
30878602
DOI:
10.1016/j.pep.2019.03.005

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