The depleted library was stored at 4°C. Affinity selection from the phage library The peptide display library was subjected to three successive rounds of affinity selection essentially as described . For selection of fusion phages from the library with IgG2a or IgA antibodies, the polystyrene Petri dish (Falcon 1007; Becton Dickinson, Lincoln Park, NJ, USA) used for panning was first coated with antibodies specific for the desired bovine immunoglobulin subclass at a concentration of approximately 20 μg/ml before the blocking step. Identification of antigens Sequences of phage displayed peptides were compared with the EMBL/GenBank database
using the BLAST programs . Flexibility, hydrophilicity, polarity and surface properties were scored using the programs Bcepred http://www.imtech.res.in/raghava/bcepred/ and BepiPred http://www.cbs.dtu.dk/services/BepiPred/[21, selleckchem 45]. Cloning, site-directed mutagenesis, expression and purification of proteins For expression, the relevant sequences of the targeted genes were amplified from genomic DNA and cloned in the pET100/D-TOPO® E. coli expression vector (Invitrogen), or in the case of PtsG, in the pQE-TriSystem His·Strep
2 vector (Qiagen). Site-directed mutagenesis (QuikChange Site-Directed mutagenesis kit; Stratagene) was used to change mycoplasmal UGAtrp codons to E. coli UGGtrp codons. Transformed E. coli cells were inoculated into Overnight Express Instant TB Selonsertib cell line medium from Novagen (Madison, learn more WI, USA). Following overnight induction, bacterial
cells were lysed using Novagen BugBuster® reagent, after which the supernatant fluids and cell pellets were analysed by SDS-PAGE and immunoblotting on a PVDF membrane using standard protocols. Proteins for PAGE analysis were purified by using ProBond nickel chelate chromatography kits as described by the manufacturer (Invitrogen). Acknowledgements We are grateful to Laurence Dedieu, François Thiacourt (CIRAD-EMVT, Montpellier, France) and Joachim Frey (Institute of Veterinary Bacteriology, University of Bern, Switzerland) for stimulating Cyclin-dependent kinase 3 discussions. We thank Jane Banda and Frances Jordaan (Onderstepoort Veterinary Institute, Republic of South Africa) for their technical help. The South African portion of this project was supported by the European Union (FP 6 INCO-DEV, Project CBPPVAC) and the General Directorate for Development and International Cooperation, French Ministry of Foreign and European Affairs (PSF No. 2003-24 LABOVET). The contribution of EMV was funded by the Wellcome Trust, London, UK, grant No. 075804. We thank Dr Philippe Totté of CIRAD for his constructive comments regarding the manuscript. References 1. Tambi NE, Maina WO, Ndi C: An estimation of the economic impact of contagious bovine pleuropneumonia in Africa. Rev Sci Tech 2006, 25:999–1011.PubMed 2.