When the peptide is cleaved, the Edans fluorophore is separated from Dabcyl, and a fluorescent signal is observed. Table 2 FRET peptide details Peptide sequence* Description d-IHSPSTGGG-e Based on CD0183 sequence d-IHGSSTPGG-e Control for above peptide d-SDSPKTGGG-e Based on CD0386, CD3392 sequence d-SDGSKTPGG-e Control for above peptide d-IHSPQTGGG-e Based on CD2768 sequence d-IHGSQTPGG-e Control for above peptide d-PVPPKTGGG-e Based on CD2831 sequence d-PVGPKTPGG-e Control for above peptide d-GQNVQTGGG-e Based on CbpA sequence d-QALPETGGG-e SaSrtA peptide d-NPQTN-e click here SaSrtB peptide d-IHSPSTGKT-e Based on CD0183 sequence d-SDSPKTGDN-e Based on

CD0386 sequence d-IHSPQTGDV-e Based on CD2768 sequence d-PVPPKTGDS-e Based on CD2831 sequence *Where d is Dabcyl (4-([4-(dimethylamino)phenyl]azo)-benzoyl) and e is Edans (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid). The N-terminal transmembrane domain of C. difficile SrtB (residues 2–25)

was replaced with a MK1775 six-histidine tag (SrtBΔN26) to improve soluble protein yield. ACP-196 mouse SrtBΔN26 was expressed in E. coli NiCo21(DE3) and purified by nickel affinity chromatography from cleared lysates (Figure 2). Purified SrtBΔN26 was then incubated with a FRET peptide containing the SPKTG sequence. An increase in fluorescence was observed over time, indicating that cleavage of the SPKTG peptide occurred in the presence of SrtBΔN26 over 48 hours (Figure 3). In addition to the SPKTG motif, SrtBΔN26 also cleaved peptides containing the predicted substrate sequences PPKTG, SPSTG, and SPQTG (Figure 4). SrtBΔN26 failed to cleave the scrambled peptide sequences GSKTP, GPKTP, GSSTP and GSQTP (Figure 4). 5FU Interestingly, SrtBΔN26 failed to cleave peptides containing the LPETG and NPQTN motifs of SaSrtA and SaSrtB, respectively, and also failed to cleave the proposed sortase recognition motif NVQTG found in the C. difficile collagen binding protein, CbpA [30] (Figure 4). Figure 2 Expression and purification of SrtB ΔN26 . E. coli NiCo21(DE3) expressing SrtBΔN26, in which the N-terminal membrane anchor has been replaced with a six-histidine

tag, were lysed by sonication and cleared lysates purified by nickel affinity chromatography. A. Anti-his western testing for expression of SrtBΔN26. Lane M: molecular mass marker, N: whole cell lysate of non-induced culture, I: whole cell lysate of culture induced with 1 mM IPTG. B. Coomassie-stained SDS-PAGE analysis of SrtBΔN26 purification over an imidazole gradient. Lane L: molecular mass marker, W: column wash, imidazole gradient indicated by grey triangle, arrows indicate the SrtBΔN26 protein. Figure 3 Cleavage of SPKTG peptide by recombinant SrtB ΔN26 . Purified recombinant SrtBΔN26 was incubated with a FRET peptide containing the SPKTG motif and fluorescence measured every hour for the first eight hours, and also at 24 h, 36 h, and 48 h.