title: Production and Purification of Recombinant Filamentous Bacteriophages Displaying Immunogenic Heterologous Epitopes.
abstract: Viruslike particles often combine high physical stability with robust immunogenicity. Furthermore, when such particles are based on bacteriophages, they can be produced in high amounts at minimal cost and typically will require only standard biologically contained facilities. We provide protocols for the characterization and purification of recombinant viruslike particles derived from filamentous bacteriophages. As an example, we focus on filamentous Escherichia coli fd phage displaying a conserved influenza A virus epitope that is fused genetically to the N-terminus of the major coat protein of this phage. A step-by-step procedure to obtain a high-titer, pure recombinant phage preparation is provided. We also describe a quality control experiment based on a biological readout of the purified fd phage preparation. These protocols together with the highlighted critical steps may facilitate generic implementation of the provided procedures for the display of other epitopes by recombinant fd phages.
authors.affiliation: Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Ghent, Belgium.|Medical Biotechnology Center, VIB, Ghent, Belgium.
authors.forename: Lei
authors.initials: L
authors.lastname: Deng
authors.affiliation: Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Ghent, Belgium.|Medical Biotechnology Center, VIB, Ghent, Belgium.
authors.forename: Florencia
authors.initials: F
authors.lastname: Linero
authors.affiliation: Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, Ghent, Belgium. Xavier.Saelens@vib-Ugent.be.|Medical Biotechnology Center, VIB, Ghent, Belgium. Xavier.Saelens@vib-Ugent.be.
authors.forename: Xavier
authors.initials: X
authors.lastname: Saelens
mesh_terms: D017104:Bacteriophage M13 / Q000187:drug effects / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D024882:Drug Resistance, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D005818:Genetic Engineering / Q000379:methods*; D015151:Immunoblotting; D013194:Staining and Labeling; D013752:Tetracycline / Q000494:pharmacology; D014170:Transformation, Genetic; D014461:Ultracentrifugation; D058425:Vaccines, Virus-Like Particle / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D004274:DNA, Recombinant; D000939:Epitopes; D058425:Vaccines, Virus-Like Particle; D013752:Tetracycline
keywords: Filamentous bacteriophage; Hybrid particles; Recombinant fd phage; Transducing units; Ultracentrifuge; pVIII coat protein