In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy

Purified, putative unprocessed and processed MBPmut-TNFa fusion proteins were analyzed by LC-MS. The average molecular weights of the eluted MBPmut-TNFa fusion proteins obtained by deconvolution of the ESI-MS spectra are given. For MBPmut-TNFa preparations from “Empty vector control” and “RseP-Myc H22F”, we found a protein eluting at 9.1 with an average molecular weight of about 45448 Da min, which was higher than expected for a MBPmut-TNFa 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 RsePMyc wt. The C-terminal sequences of the substrate MBPmut- … CPFLSLFSFL39 fusion protein and processing product MBPmut- … CPFLSLF36 were confirmed by LC-MS/MS analysis.