Figure 2
Recombinant expression and purification of REAMP2.0. (a) REAMP2.0 was isolated from E. coli cellular membranes by affinity chromatography. Cell fractions boxed in the workflow are retained and analysed by Coomassie-stained SDS-PAGE and anti-V5 western blot (IB a-V5). The theoretical molecular weight of the StrepII-tagged REAMP2.0 is 22.9 kDa. The uncropped western blot is provided as Figure S3. (b) Size exclusion chromatography, (c) static light scattering and (d) native nanoelectrospray mass spectrometry all confirm that purified REAMP2.0 is a homogenous, monodisperse monomer in the solubilising detergent Cymal-5. (e) Solvent extracts of cell membranes from induced strains of REAMP2.0H accumulate a novel pigment when supplemented with the heme precursor δ-aminolevulinic acid (ALA). Treatment controls shown include uninduced (-IPTG) and unsupplemented (-ALA) strains. (f) fluorescence spectra of membrane extracts confirm the pigment as zinc protoporphyrin IX by reference to a commercial standard. (g) REAMP2.0H expression correlates with cellular zinc porphyrin. (h) Ratio of absorption peaks from ZnPPIX (A420) and heme (A401) in membrane solvent extracts. + Fe, culture media with 0.1 mM ammonium iron sulfate. CybB, strain overexpressing recombinant E. coli diheme cytochrome CybB. Data in (g) and (h) are mean ± SD of 3 independent repeats.
