Figure 2

Identification of DevS binding peptides using biopanning. (A) Enrichment of phages binding to DevS protein. Equal numbers of phage particles (10¹¹) before (unpanned library) and after three rounds of biopanning on DevS (Pan 3) were incubated in wells coated with DevS or BSA or left uncoated. The unbound phages were removed by washing the wells with TBS containing 0.5% Tween 20 and the bound phages were detected with HRP-conjugated anti-M13 antibody (Amersham) and o-Phenylenediamine substrate. The assays were performed in triplicate and the mean ± SD values are plotted. (B) Phage ELISA of selected phage clones. Phage clones displaying DevS-binding peptides were used in ELISA. Equal numbers of phage particles after amplification were incubated in wells coated with DevS or BSA or left uncoated. The bound phages were detected as described above. (C) Alignment of peptides identified by DevS panning with the sequence of DevR. Functionally important and conserved residues: D54, phosphorylation site; T82, K104, residues involved in phosphorylation induced conformational changes, are highlighted in grey. Screening a phage display library for DevS binders identified the 'Pep’ peptides. DevRS peptides were designed on the basis of DevR sequence. CLUSTALW-based alignment of the 'DevRS’ peptides with the DevR sequence is shown. *, positions having a fully conserved residue; :, conservation between groups of strongly similar properties; ., conservation between groups of weakly similar properties. DevRS B-ext could not be analyzed due to the inability to prepare soluble peptide.