Fig. 4. C11 family proteases of P. dorei activate BcpT.

a Left panel: Coomassie-stained gel of purified recombinant doripains (Dpns) and their ability to cleave BcpT. Purified recombinant doripain A (DpnA) incubated with trypsin at a 1:50 (w:w) trypsin to DpnA ratio for 15 mins at 37 °C was sufficient to cleave the activation loop without degradation of the protein. DpnA activated in this manner is referred to as DpnA(a). Red boxes indicate cleaved doripain B (DpnB) fragments that eluted with contaminating proteins from E. coli, possibly due to their association with DpnB. Right panel: FXa and DpnB cleaved BcpT showing the same sized fragments are generated. Arrows indicate the DpnB protease fragments that result from its activation b Agar overlay assays showing that the Dpns can activate BcpT like FXa and inhibit the growth of Pv8482. Dpns did not activate the R65N or the R199N-R65N double mutants, however, a weak activation of the R199N was observed. Although this assay is only semi-quantitiative, the spot of the undiluted R199N (5 μg) most closely resembled that of the 0.04 μg spot of the WT, which is <10% of WT activity. c Western immunoblot of purified BcpT and site mutants of BcpT cleaved by DpnA(a) and DpnB. The blot was probed with α-BcpT. Note that the smaller fragment in the R65N in the DpnA is smaller than that of the DpnB cleaved R65N mutant. We have found that DpnA cleaves the His-tag more efficiently than DpnB which accounts for the difference in size of the small fragment of ~2.5 kDa. d Western immunoblot analysis of BcpT cleavage products in the supernatant of PdCL02 and dpn mutants and complemented mutants. e Agar overlay assay analyzing the ability of PdCL02 dpn mutants to inhibit the growth of PvCL10. Overlays and BcpT cleavage analyses were performed two to three times with consistent results. All gels and blots were perfomred a minimum of two times with consistent results. Source data are provided as a Source Data file.