The number below each of the proteases in larger see more font is the calculated pI of the respective mature protease. Table 1 Identity and similarity matrix for Bacteroides C10 proteases   SpeB Bfp1 Bfp2 Bfp3 Bfp4 BtpA BtpB BtpC BtpZ SpeB   21.6 a 18.0 22.6 23.3 22.6 20.0 21.6 21.1 Bfp1 43.0 b

  22.4 25.1 21.1 22.8 21.7 19.4 19.3 Bfp2 35.1 38.6   20.3 22.9 26.5 20.2 22.5 18.3 Bfp3 42.2 46.5 40.0   29.0 27.6 23.5 25.2 21.0 Bfp4 43.5 44.4 41.2 49.1   27.4 22.7 22.4 22.3 BtpA 42.3 45.8 44.1 49.8 49.3   22.4 21.9 22.8 BtpB 38.4 38.4 40.4 39.9 42.6 39.9   54.3 27.7 BtpC 38.4 39.0 41.9 43.6 44.5 40.6 72.5   29.2 BtpZ 41.3 40.6 41.2 41.5 45.4 42.3 47.3 47.7   a Numbers in bold indicate percentage identity. b Numbers in italics indicate percentage similarity.

Figure 2 Sequence relationship for C10 proteases from B. fragilis , B. thetaiotaomicron and S. pyogenes . (a) learn more Cladogram constructed for C10 protease sequences. (b) Amino acid sequence alignment of Btps from B. thetaiotaomicron with catalytic residues in SpeB. The four predicted proteases, BtpA, BtpB, BtpC SN-38 nmr and BtpZ, had deduced molecular mass values of 48714 Da, 52555 Da, 51669 Da and 47549 Da respectively, making them significantly larger molecules than SpeB (43174 Da). Similar to the Bfp enzymes, all four predicted proteases in B. thetaiotaomicron included an amino-terminal export signal with a cleavage site for signal peptidase II, suggesting they are lipoproteins. These leader peptides spanned residues 1–17 for BtpA, BtpB and BtpC and residues 1–19 for BtpZ. Proteases are typically expressed with a propeptide which promotes proper folding, and prevents inappropriate proteolytic activity. Guided by sequence comparisons with SpeB, all Btp proteins included primary sequence consistent with an N-terminal propeptide (residues 18–154, 18–201, 18–196 and 20–159 for BtpA, BtpB, BtpC and BtpZ respectively, and indicated in Figure 1). Also of note is the acidic nature of the clustered Avelestat (AZD9668) gene products BtpB, BtpC and BtpZ with pI values of 5.25, 5.05 and 4.80 for the predicted mature form of the proteins. This contrasts

with the basic values of 9.22 for BtpA and 8.49 for SpeB. Sequence alignment and secondary structure predictions for the predicted proteins (BtpA, BtpB, BtpC and BtpZ) with the B. fragilis and S. pyogenes orthologues supports the idea that these proteases adopt a papain-like fold (data not shown). The catalytic Cys and His residues were conserved in BtpA, BtpB and BtpC (Figure 2(b)), though interestingly, the catalytic Cys residues was not preserved in BtpZ. As noted above, co-expression with small, genetically linked protease inhibitors has emerged as a common theme for bacterial papain-like proteases [9, 20, 22]. Three candidate inhibitor genes were identified in open reading frames (ORFs) adjacent to the btp genes in B.