The purification of the σ FpvI /FpvR 20 and σ PvdS /FpvR 20 protein complexes is facilitated at room temperature.

Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σ ^FpvI and σ ^PvdS 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 σ ^FpvI and σ ^PvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR ₂₀ and the σ ^FpvI and σ ^PvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σ ^FpvI and σ ^PvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His ₆ -σ ^FpvI /FpvR _1-67 , His ₆ -σ ^FpvI /FpvR _1-89 , His ₆ -σ ^PvdS /FpvR _1-67 and His ₆ -σ ^PvdS /FpvR _1-89 protein complexes (where FpvR _1-67 and FpvR _1-89 are truncated versions of FpvR ₂₀ ) 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 His ₆ -σ ^FpvI /FpvR _1-67 and His ₆ -σ ^PvdS /FpvR _1-67 complexes. A comparison of these values with those previously reported for the His ₆ -σ ^FpvI /FpvR _1-89 and His ₆ -σ ^PvdS /FpvR _1-89 complexes is made.
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