Fig. 3: Visualization of in vivo intramembrane proteolysis catalyzed by RseP-Myc. | Communications Biology

Fig. 3: Visualization of in vivo intramembrane proteolysis catalyzed by RseP-Myc.

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

Fig. 3

a E. coli T7 Express cells harboring the two compatible plasmids pETDuet-1 and pCOLADuet-1, which co-express genes encoding the heme-binding protein MBPmut-TNFα-(1-39)-L31P and the active RseP-Myc wt or inactive RseP-Myc H22F protease, were incubated for five days at room temperature on an agar plate. Control cells expressed only one gene encoding either RseP-Myc wt, RseP-Myc H22F, the substrate MBPmut-TNFα-(1-39)-L31P or the putative proteolysis product MBPmut-TNFα-(1-34)-L31P. Green E. coli colonies are only observed if the substrate present, MBPmut-TNFα-(1-39)-L31P, is not C-terminally truncated by RseP. b and c Analysis of E. coli T7 Express cells expressing genes for RseP-Myc wt, RseP-Myc H22F, MBPmut-TNFα-(1-34)-L31P, MBPmut-TNFα-(1-39)-L31P or coexpressing genes for MBPmut-TNFα-(1-39)-L31P and RseP-Myc wt/H22F, respectively, and grown on a selection plate for four days at room temperature by b, SDS-PAGE, and c, by immunoblotting using a monoclonal anti-Myc-Tag antibody. Comparative in vivo analysis of RseP activities as shown in a and b was repeated with untagged RseP proteases with the same result (compare Supplementary Fig. 2).

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