Recent advances in biotechnology have enabled the development of innovative approaches for targeted delivery of biomolecules, among which recombinant vaccines and gene therapy are particularly prominent. Filamentous bacteriophages represent a promising platform for surface display of antigens, as they allow stable incorporation of fusion proteins into capsid structures, thereby enhancing immunogenicity and enabling efficient targeted delivery. Owing to their simple genetic and protein engineering as well as their ability to effectively present antigens, filamentous bacteriophages are increasingly explored as delivery systems for recombinant vaccines. In this master’s thesis, the SnoopTag/SnoopCatcher bioconjugation system was employed for the indirect attachment of a model antigen to the bacteriophage capsid. This system is based on the specific and irreversible formation of an isopeptide bond between two complementary fragments derived from the D4 domain of the adhesin RrgA from Streptococcus pneumoniae. Such an approach enables precise and stable protein conjugation without the need for chemical modifications or additional reagents, which is important for preserving the biological functionality of antigens. The aim of this work was to develop and evaluate a platform for antigen display on the surface of filamentous bacteriophages using the SnoopTag/SnoopCatcher interaction. For this purpose, the plasmid pET-28a carrying the gene encoding the fusion protein SnoopCatcher–sfGFP was introduced into the expression strain Escherichia coli (E. coli) NiCo21 (DE3). After expression, the recombinant protein was isolated and purified by immobilized metal affinity chromatography, and its purity was assessed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). In parallel, the gene encoding SnoopTag-cMyc fused to the capsid protein pVIII was inserted into the phage vector f88KE. The efficiency of SnoopTag/SnoopCatcher conjugation was analyzed by SDS-PAGE and Western blot analysis and confirmed using antibodies against cMyc, His, and GFP, which specifically recognize individual parts of the fusion protein and the resulting conjugate. Quantitative evaluation of conjugation was performed by densitometric analysis of luminescent signals in comparison with reference proteins of known quantities. The results demonstrate that the SnoopTag/SnoopCatcher system enables stable conjugation of a model antigen to the surface of filamentous bacteriophages. Such phage constructs represent a promising platform for the development of recombinant vaccines and immunotherapeutic strategies, and may also serve as a basis for biosensors in advanced molecular diagnostic applications.
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