Fig. 4: Characterization of the RseP cleavage product in vitro.
a Purification of membrane-associated MBPmut-TNFα fusion proteins by amylose affinity chromatography from IPTG induced T7 cells co-expressing either genes for MBPmut-TNFα-(1-39)-L31P and RseP wt (left column) or genes for MBPmut-TNFα-(1-39)-L31P and mutant RseP H22F (right column). The purified MBPmut-TNFα fusion proteins (which correspond to the product and substrate of the RseP reaction, respectively) were then analyzed b by SDS-PAGE, c by UV/Vis spectroscopy [blue trace, + RseP wt; orange trace, + RseP H22F; showing the full spectra (left absorbance axis) and the same spectra magnified (right absorbance axis)] and d by gel filtration on a Superose 6 Increase column [mAU = milli absorbance units; absorbance is monitored at 280 nm (blue trace), 371 nm (orange trace), and 451 nm (grey trace)]. As expected, the heme binding dimers and processing products (indicated by arrows) show different A371nm/A451nm absorbance ratios. It can be assumed that heme binding to monomeric TNFα processing products significantly alters their shape/structure and thus their elution volume and apparent molecular weight. e UV/Vis spectroscopy of gel filtration fractions containing heme binding MBPmut-TNFα fusion proteins (blue trace, + RseP wt, elution volume 15.4 ml – 16.2 ml; orange trace, + RseP H22F, elution volume 15.4 ml – 15.8 ml of the gel filtration runs shown in Fig. d). Purification of MBPmut-TNFα proteins coproduced with either RseP wt or with RseP H22F (a-c) was performed four times and representative results are shown. b-e belong to the same experiment. Gel filtration (d and e) of the purified MBPmut fusion proteins was performed once.
