Coiled-coil protein origami (CCPO) is one of the de novo approaches to protein design, which is based on modularity and long-range interactions between the individual segments of a protein. So far, protein origami smaller than 100 kDa in the form of tetrahedra, tetrahedral pyramids, bipyramids and trihedral prisms have been successfully designed and characterised. When designing even larger protein origami of a few 100 kDa, the main hurdle is the limited size of our set of orthogonal coiled-coils that are the foundation of the CCPO. The aim of this MSc thesis was to develop a new strategy for the design of larger structures based on already developed structures of CCPO and the ability of protein domain presentation at their vertices. We upgraded the protein origami in the form of the TET12SN tetrahedron by integrating the SpyCatcher/SpyTag system, which has the ability to form isopeptide bonds. Initially, we designed the basic building blocks of the system by decorating the vertices of TET12SN with one, two, three or four SpyCatcher or SpyTag domains. All constructs decorated with the SpyCatcher protein domain retained the same catalytic activity as the free SpyCatcher domain, whereas SpyTag domains at the same insertion sites did not retain activity. Therefore we experimentally identified optimal sites for the insertion of the SpyTag protein domain with respect to activity retention, and thus designed TET12SN decorated with one, two, three or four SpyTag domains. After successful isolation and characterisation of all the basic constructs by analytical size exclusion chromatography, circular dichroism spectroscopy, small-angle X-ray scattering and native polyacrylamide gel electrophoresis, we proceeded to the formation of complex structures. Here, we combined different basic constructs, using a basic construct with a different number of SpyCatcher or SpyTag domains as the central scaffold and a basic construct with a single copy of a suitable pair, SpyTag or SpyCatcher, as the ligand scaffold. The basic building blocks were linked via isopeptide bonds and the resulting complexes were characterised by analytical size exclusion chromatography, native polyacrylamide gel electrophoresis, sodium dodecyl-sulfate polyacrylamide gel electrophoresis and small-angle X-ray scattering by which we successfully confirmed the formation and shape of mono-, di-, tri- and tetraconjugated complexes. The complexes designed in this way are the largest protein origami described to date, with the smallest such characterised conjugated complex having a molecular mass of 170 kDa and the largest one exceeding 400 kDa. The system developed in the course of the MSc thesis could be further upgraded by the integration of other components (e.g. nanobodies, antigens, enzymes or other binding domains), thus taking a step closer to the functionalisation of protein origami for medical or industrial applications.
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