Fig. 5: Determination of RseP cleavage sites.

a and b Membrane-associated MBP_mut-TNFα fusion proteins from IPTG-induced E. coli T7 Express cells were purified by amylose affinity chromatography and analyzed by SDS-PAGE. a MBP_mut-TNFα-(1-39)-L31P was co-expressed with pCOLADuet-1-rseP wt (R) or the empty vector control pCOLADuet-1 (V), or b with pCOLADuet-1-rseP-Myc wt (wt) or pCOLADuet-1-rseP-Myc H22F (H22F). Comparable amounts of purified proteins were run twice and alternately on SDS-PAGE to detect very small differences in molecular weight. c UV/Vis spectra of MBP_mut-TNFα fusion proteins purified from these different genetic backgrounds (orange trace, empty vector control; magenta trace, + RseP wt; blue trace, + RseP-Myc H22F; green trace, RseP-Myc wt). d Calculated average molecular weights of C-terminally truncated MBP_mut-TNFα-(1-39)-L31P fusion proteins (only the C-terminal residues of the MBP_mut-TNFα fusion proteins are shown here and in e). e Purified, putative unprocessed (left panels) and processed (right panels) MBP_mut-TNFα fusion proteins (compare a and b) were analyzed by LC-MS. The average molecular weights of the eluted MBP_mut-TNFα fusion proteins obtained by deconvolution of the ESI-MS spectra (compare Supplementary Fig. 4a-d) are given. The substrate MBP_mut- … CPFLSLFSFL³⁹ and the degradation product MBP_mut- … CP³¹ (found in all samples) are shown in grey, RseP major processing products MBP_mut- … CPFLSLF³⁶ and MBP_mut- … CPFL³³ in brown and additional processing products in black font. For MBP_mut-TNFα preparations from “Empty vector control” and “RseP-Myc H22F”, we found a protein eluting at 9.1 min with an average molecular weight of about 45448 Da min, which was higher than expected for a MBP_mut-TNFα fusion protein and was therefore not further characterized. Compounds eluting at retention times above 9.5 min (corresponding to 90% acetonitrile) were contaminants not further characterized. a to e belong to one experiment, and the processing specificity of RseP wt was confirmed in this experiment by analysis of RseP-Myc wt. The C-terminal sequences of the substrate MBP_mut- … CPFLSLFSFL³⁹ fusion protein and processing product MBP_mut- … CPFLSLF³⁶ were confirmed by LC-MS/MS analysis (Supplementary Fig. 4e).