The use of filamentous bacteriophages for the development of recombinant modular vaccines is considered a promising platform due to their favorable characteristics, such as easy genetic modification, high specificity (and thus safety), immunogenicity, and high stability. So far, in vitro/vivo research has demonstrated positive anti-cancer, anti-viral, anti-parasitic, and anti-bacterial effects of antigenic peptide display bacteriophage vaccines. However, the improper folding of the presented antigenic proteins, their ineffective transfer to the periplasm, and steric hindering of capsid assembly which all lead to an insufficient display level severly hamper the development of phage-based vaccines. Alternatively, short peptides from split proteins for protein antigen payload tethering might be well-suited for phage display. SpyTag:SpyCatcher is an example of split protein system in which the two components form a covalent (isopeptide) bond upon selective binding.
In the scope of the master's thesis, we prepared four genetic constructs for the SpyTag-capsid protein 8 fusions, which were inserted into the vector f88KE (a type 88 phage display vector harboring 2 copies of of the major capsid protein pVIII, a wild-type and a recombinant one). The constructs differed in combinations of the signal peptide (directing the periplasmic transfer either co- or posttranslationally) and the version of the SpyTag peptide (SpyTag002_T112H or SpyTag003) having slightly different physico-chemical properties. In the fifth genetic construct, we introduced rare codons for leucine in the wild-type copy of the gene encoding protein pVIII. We speculated that this would lead to reduced expression of wild-type pVIII and consequently more recombinant fusion protein pVIII copies would be integrated in the capsid (in one of the designed phage vectors such constructs were combined). Using ELISA and western blot assays, we comparatively evaluated the display levels of peptides. We found that rare leucine codons do not significantly reduce the expression of wild-type pVIII, nor does the combination of genetic constructs improve the presentation of the SpyTag-pVIII recombinant protein on the filamentous bacteriophage capsid. However, we observed significant differences in display levels of SpyTag peptides depending on the type of signal peptide and SpyTag variant's positive charge. The best combinantion included the signal peptide directing proteins to periplasm co-translationally and the SpyTag002_T112H peptide variant having a slightly lower positive charge.
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