Optimisation of the purification process of Potato virus Y virus-like particle and development of methods for its functionalisation and super-resolution imagingTomsič, Nika (Avtor) de Marco, Ario (Mentor) Dolinar, Marko (Komentor) virus-like particlepotato virus Ypurification optimisationSpyTag-SpyCatcher systemnon-canonical amino acidsNanotechnology plays an important role in medicine. One of the most significant advancements in this field is the use of nanobodies since they represent a major advantage over classical antibodies. The main advantages are their small size (15 kDa), effective biodistribution and short circulation time, elevated tissue penetration, stability, solubility and biocompatibility. Also, virus-like particles (VLPs) are often used in nanotechnology. These are non-infectious virus particles because they do not contain viral genetic material. In medicine, they have been exploited for targeted drug delivery, as vaccines and in immunotherapy. An interesting example is the Potato virus Y, the main pathogen of potatoes. Mutations of its coat protein induce the formation of VLPs of an eight-ring cubic structure. The aim of the thesis was to optimize the purification protocol for the Potato virus Y coat protein fused to SpyTag on its C-terminal and to prove that the corresponding purified cubic VLPs can be functionalized with nanobodies using the SpyTag-SpyCatcher system. This would serve as the proof of concept for the use of such structures in medical applications, for example in pathogen targeting. Additionally, the incorporation of 4 azido-L-phenylalanine (4AZF) into nanobody sequences for direct conjugation with a dye was attempted to prepare reagents for super-resolution microscopy. The purification protocol was optimized by combining size exclusion and anion exchange chromatography. The binding of nanobodies onto VLPs was proved, but its efficiency remained low, and the complex was below the detection limit of native agarose gel electrophoresis. It is unclear if this was due to the low accessibility of SpyTag on the VLP’s structure. The construct in which the 4AZF was incorporated was successfully prepared using the following conditions: a) the amber codon was inserted between the nanobody sequence and the sequences of 6×His tag and AviTag, b) sulfhydryl oxidase was co-expressed to allow correct nanobody folding; c) specific tRNA, tRNA synthetase, and synthetic non-canonical amino acid (4AZF) were present when protein expression was induced. 20242024-10-25 12:30:02Magistrsko delo/naloga164453VisID: 24246COBISS_ID: 220010499sl