br Experimental design materials and methods Secondary struc
Experimental design, materials and methods Secondary structure analysis of diluted HpFlgD (2mgmL−1) was performed by circular dichroism (CD) using a spectropolarimeter (Jasco Analytical Instruments) in the far UV region (190–260nm), Fig. 1. Afterwards, the data were deconvoluted using software CDNN  and are shown as contributions of the various components to the protein secondary structure (Table 1). The level of degradation of HpFlgD_26695 and crystallized HpFlgD_26695 was monitored by the SDS-PAGE. The sample from the crystal of the tetragonal form of HpFlgD_26695 (Fig. 2b and c) was prepared by dissolving the crystal in the SDS-PAGE loading buffer. This sample together with a full length HpFlgD_26695 was checked by SDS-PAGE (Fig. 3a). The bands obtained from the crystallized sample and full length HpFlgD_26695 were isolated and in gel digested with trypsin. The fractions of the extracted peptides were dried out, dissolved in 50% acetonitrile, supplemented with 0.1% formic retinoid x receptor and directly injected in the nano-ESI source. Mass measurements were performed with a quadrupole-TOF spectrometer (Waters, Manchester, UK) (capillary voltage: 2800–3000V; cone voltage: 45V; scan time: 1s; interscan: 0.1s). Analysis of the spectra was performed by using the MASSLYNX software (Micromass, Wynthenshow, UK). The data obtained from the mass analysis are presented in Fig. 4. The mass of the HpFlgD_G27 monomer was determined by mass analysis of the peaks isolated by reverse phase chromatography (C4-column, RP-HPLC), Fig. 5. Presence of the His tag at the C-terminus of the full length HpFlgD_26695 and crystallized HpFlgD_26695 was evaluated with anti-His antibodies (Mouse monoclonal, 1:1000 dilution) and secondary antibodies (Goat anti-mouse HRP, 1:10,000) (Western blotting technique), Fig. 3b. Fig. 6 shows different types of interfaces present in both crystal forms of HpFlgD. In Table 2 the interface area, the number of hydrogen bonds and salt bridges involved in each interface are shown. The list of hydrogen bonds responsible for the tetramerization is presented in Table 3. Superposition of the Fn-III domain in HpFlgD with the fibronectin domain in 1FNA  is presented in Fig. 7, while the superposition of the tudor domain in HpFlgD and the same domain in PaFlgD (PDB ID: 3OSV, ) and XcFlgD (PDB ID: 3C12, ) can be seen in Fig. 8. Fig. 9 presents the overlayed structures of HpFlgD and modeled HpFlgE. Modeled HpFlgE was prepared by homology using software Phyre2.
Acknowledgments This work was supported by the University of Padua, by PRIN 2010–2011 (MIUR) “Unraveling structural and functional determinants behind Helicobacter pylori pathogenesis and persistence”.
Value of the data
Data Fig. 1 shows the SDS-PAGE analysis comparing soluble and insoluble protein content for the proteins LovR and SHY2 tagged with two different protein tags and expressed at different conditions. Additionally, the electrophoretic analysis in Fig. 2 compares the content of inclusion bodies. Recombinant proteins tagged with the protein tags CusFp and SmbPp, containing their signal sequences, are exported to the cell׳s periplasm. Fig. 3 shows an image of E. coli BL21(DE3) cells after RFP expression and the electrophoretic analysis of the periplasmic lysates. Fig. 4 shows pictures of the synthesized silver chromatographic media before and after incubation with the E. coli lysate expressing green fluorescent protein tagged with CusF. Fig. 5 shows the SDS-PAGE analysis of the purification steps, it shows the protein content in the flow-through (the lysate after incubation with the Ag(I) resin), and two elutions steps with 160mM methionine.
Experimental design, materials and methods
Acknowledgments We thank Mexico׳s Consejo Nacional de Ciencia y Tecnologia (CONACYT) for financial support provided to JECB and TVC during their graduate studies. This work was funded by CONACYT Grant CB 2012-179774-B awarded to XZ.