Purification of the Campylobacter jejuni Dps protein assisted by its high melting temperature

Highlights

• We determined that C. jejuni Dps has an unusual high melting temperature.

• We report a simple and reproducible protocol C. jejuni Dps purification.

• Gel filtration and circular dichroism confirmed that the C. jejuni Dps structure was preserved.

• DNA-binding analysis confirmed the functionality of the purified C. jejuni Dps.

Abstract

Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe²⁺ in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe³⁺, is then stored inside the protein shell in the form of non-reactive insoluble Fe₂O₃. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characterisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected.

Introduction

Dps proteins are highly conserved and widely distributed iron storage proteins present in a vast range of prokaryotes. Dps proteins form a distinct group within the ferritin superfamily. The crystallographic structure of various Dps members has been solved; in all cases they are composed of 12 identical subunits (of approximately 150–250 residues) that are assembled into a conserved spherical protein shell [1], [2], [3].

The central core of the Dps dodecamer contains conserved residues that form the ferroxidase center were Fe²⁺ is oxidized to Fe³⁺ and then stored inside the protein shell as insoluble and non-reactive Fe₂O₃ [4], [5]. The Dps ferroxidase center is the most remarkable signature of the Dps family, it is located at the interface between monomers related by 2-fold symmetry [6]. Dps can either use hydrogen peroxide or oxygen to facilitate Fe²⁺ oxidation [7]. The concomitant removal of iron and hydrogen peroxide inhibits Fenton chemistry (H₂O₂ + Fe²⁺ → OH⁻ + OH* + Fe³⁺) thereby relieving the formation of toxic hydroxyl radicals.

Besides iron storage and hydrogen peroxide detoxification, some Dps members perform other cellular functions, such as DNA binding and protection [8], [9], resistance to various forms of stress [10], [11], [12], [13], carbohydrate binding and cell adhesion [14] as well as promotion of bacterial survival within biofilms [15], [16].

Campylobacter jejuni is one of the most common causes of bacterial gastroenteritis worldwide. Infection by C. jejuni results in rapid onset of fever, abdominal cramps and diarrhea [17], [18]. The disease is usually self-limiting; nonetheless complications such as reactive arthritis and the neuroparalytic Guillain–Barré syndrome have been associated with C. jejuni infections [19]. Multiple functions have been ascribed to C. jejuni Dps (CjDps)¹ protein such as DNA binding and protection, survival in response to hydrogen peroxide and binding to the galactoceramide sulfatide [20]. In vivo studies showed that CjDps is important to biofilm formation and caecal colonization in poultry. Furthermore, preliminary studies demonstrated the potential of using CjDps protein as a vaccine antigen in chicks [15].

In order to facilitate full functional characterization of CjDps, it is important to have a simple and reproducible protocol for protein purification. Several studies have reported the expression and purification of recombinant CjDps. In all cases, a His-tag has been introduced either at N or C-terminal to facilitate protein purification by affinity chromatography [15], [21], [22]. As Dps exist as dodecamer complex, the presence of His tags in each of its monomers may alter the overall Dps surface. It has been reported that Dps from different organisms can bind DNA in a pH-dependent manner, probably through electrostatic interactions [9], [21], [23]. The presence of histidines, which are particularly sensitive to protonation at near physiological pH, might influence the overall Dps charge and thus its DNA binding properties. It is therefore imperative to be able to purify this protein without a tag to enable natural dodecamer formation and accurate functional characterization. Here, we report a purification protocol for the untagged C. jejuni Dps protein.