Nanomaterials have long been explored as efficient systems for delivering target molecules into cells. Synthetic nanocarriers have several design and manufacturing limitations, making natural nanocarriers such as virus-like particles (VLPs) are a better alternative. VLPs are nano-sized and mimic the structure of viral particles without the viral genome. As such, VLPs are a promising platform for vaccine design. The SpyCatcher:SpyTag split protein system originates from the CnaB2 domain of the Streptococcus pyogenes fibronectin adhesion protein FbaB and facilitates simple conjugation of proteins, including VLPs. SpyCatcher consists of 138 amino acid residues and spontaneously forms an isopeptide bond with the 13-amino acid peptide SpyTag, and together they form a covalently bound complex. Scientists have developed the second (SpyCatcher002, SpyTag002) and third generation (SpyCatcher003, SpyTag003) of this split protein system based on the phage display technique and crystal structure-guided rational design, respectively. These have improved interaction kinetics and are more resistant to temperature changes. In this Master’s thesis, we have expressed in Escherichia coli the SpyCatcher003-mi3 fusion protein, which forms a stable nanocage, while SpyTag003 was fused to a monomeric red fluorescent protein. The recombinant protein SpyTag003-mRFP was isolated for further analysis by immobilized metal affinity chromatography and the SpyCatcher003-mi3 was precipitated from bacterial lysate and then purified by size exclusion chromatography. Purity and homogeneity were determined by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, and the concentration of the expressed proteins was determined by the Bradford method. Expression of both fusion proteins was successful, yielding highly pure SpyCatcher003-mi3 and SpyTag003-mRFP, which were fused by conjugation reaction in a 1:1.5 molar ratio in favour of SpyTag003-mRFP. The conjugation reaction was evaluated against the negative control of a mixture of SpyCatcher003-mi3 and AP4-mRFP (which do not form a conjugate). Using the dynamic light scattering method, we demonstrated that the protein conjugation was successful and a monodisperse and homogeneous mi3-SpyCatcher003:SpyTag003-mRFP nanoparticle conjugate was obtained. In an experiment with immortalised mouse macrophages, we evaluated the success of phagocytosis of nanoparticles. Based on the results, it can be concluded that the SpyCatcher:SpyTag system is a simple and efficient platform for the production of novel nanoparticle-based vaccines. The development and use of VLPs combined with recombinant antigens could contribute to overcoming major challenges such as rapidly evolving pathogens or zoonotic outbreaks.
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