Casas Garcia G Patricia, Perugini Matthew A, Lamont Iain L, Maher Megan J
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Protein Expr Purif. 2019 Aug;160:11-18. doi: 10.1016/j.pep.2019.03.005. Epub 2019 Mar 13.
Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σ and σ 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 and σ sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR and the σ and σ proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σ and σ 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-σ/FpvR, His-σ/FpvR, His-σ/FpvR and His-σ/FpvR protein complexes (where FpvR and FpvR are truncated versions of FpvR) 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-σ/FpvR and His-σ/FpvR complexes. A comparison of these values with those previously reported for the His-σ/FpvR and His-σ/FpvR complexes is made.
细菌含有σ因子，可响应各种环境刺激来控制基因表达。替代σ因子σ和σ特异性结合抗σ因子FpvR。这些蛋白质是铜绿假单胞菌基于绿脓菌素的铁摄取系统的重要组成部分。由于该系统的独特性，即σ和σ这两种σ因子的活性均由同一抗σ因子调节，因此抗σ蛋白FpvR与σ和σ蛋白之间的相互作用已在体内得到广泛研究。然而，难以获得可溶性的重组σ和σ蛋白制剂，这限制了它们的生化和结构表征。在本研究中，我们描述了一种纯化方案，该方案能够以高纯度和高浓度生产可溶性重组His-σ/FpvR、His-σ/FpvR、His-σ/FpvR和His-σ/FpvR蛋白复合物（其中FpvR和FpvR是FpvR的截短版本），适用于通过圆二色光谱和分析超速离心进行生物物理分析。这些结果表明蛋白质在溶液中折叠，并导致测定His-σ/FpvR和His-σ/FpvR复合物内蛋白质-蛋白质相互作用的亲和力。并将这些值与先前报道的His-σ/FpvR和His-σ/FpvR复合物的值进行了比较。
