Synthetic biology engineering to catalyse unbreakable linkage between peptide building blocks

Protein conjugation is an important tool for research and the development of novel therapeutics. Peptide tags are ideal handles for bioconjugation, as they are minimally disruptive to protein structure and function and are weakly immunogenic. In this thesis, I have developed SnoopLigase, a protein which is able to covalently link the two peptide tags SnoopTagJr and DogTag by site-specific transamidation. The three modules were obtained by splitting an isopeptide bond-containing domain of the Streptococcus pneumoniae adhesin RrgA. Using rational and computational protein design, rate and yield of the reaction were improved, while allowing efficient reaction in different buffers and temperatures. The separation of conjugated proteins from unreacted substrate can be challenging when using established bioconjugation techniques. SnoopLigase bound strongly to its reaction product, which enabled the purification of conjugated SnoopTagJr-DogTag by SnoopLigase solid-phase immobilisation and subsequent product elution. Hence, SnoopLigase could be useful to produce high quality bioconjugate therapeutics. Vaccines are important medical interventions, saving millions of lives each year. Recombinantly produced antigens are cost-effective and have an unmatched safety profile, yet often show poor immunogenicity. Protein multimerisation can strongly enhance the immune response against the antigen. Based on the SnoopLigase system and the heptameric protein IMX313, I developed a highly stable, modular vaccine platform. Covalent conjugation of SnoopTagJr-linked proteins to the platform nanoparticle IMX313-DogTag allowed simple, efficient and modular protein multimerisation. The efficient coupling by SnoopLigase and the small size of the tags provide potential to accelerate development of modular vaccines against malaria and other diseases.