Pet16b plasmid

Epitopes recognised by the MAbs were mapped using a set of truncated PLY fragments. For the epitope mapping, PLY was divided into eight overlapping truncated protein fragments (Table 1).
These fragments were synthesised in E. coli, as described previously [33 (link)]. Briefly, the DNAs containing the coding sequences of the PLY fragments were amplified by PCR. The primers (Metabion International AG, Planegg, Germany) used to generate PLY1–PLY8 fragments are listed in Table 2. The amplified DNA fragments were verified by sequencing, cloned the pET16b plasmid (Merck KGaA), and the resulting plasmid was transformed into E. coli Tuner (DE3) strain (Merck KGaA). After induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), the synthesis of recombinant N-terminally hexahistidine tagged protein fragments was confirmed both by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and Western blot (WB) using anti-His-Tag MAb (MA1-21315, Thermo Fisher Scientific). The reactivity of the MAbs with the truncated PLY fragments was analysed by WB.

DTT, BSA and antibiotics were purchased from Melford Laboratories; polyacrylamide-bis polyacrylamide [30% (w/v), 37:5:1], Bacto tryptone and yeast extract for culture media were purchased from Oxoid. Chelating fast flow resin and Superdex 200 resin were purchased from GE Healthcare; primers were purchased from Eurofins; restriction enzymes and E. coli strain K12 JM109 were purchased from New England Biolabs; pET16b plasmid was purchased from Merck Chemicals; E. coli RelA was expressed using a strain from the ASKA Clone library purchased from Shigen; E. coli MRE600 (C6) strain was purchased from NCTC. Francisella philomiragia was obtained from A.T.C.C.; mRNA was purchased from ATDBio. Unless stated otherwise all other reagents were purchased from Sigma–Aldrich or Fisher Scientific. Graphpad Prism version 6 for Windows was obtained from Graphpad Software.