TY - GEN A1 - Jaut, Valerie T1 - Investigation of the engineering of Leishmania RNA Virus 1 capsid protein T2 - Masterarbeit N2 - Virus-like particles are of great interest. Due to the lack of functional amino acids, they are non-infectious and are widely investigated for their usage in the biomedical field, such as nanomaterials, vaccines, drug delivery. This thesis is about the engineering of virus-like particles, which are formed by self-assembly of LRV1 capsid proteins. Basis was a publication by Procházková et al. (2021), where the structure of virus-like particles, derived from recombinant LRV1 capsid proteins, was determined using cryo-electron microscopy. Goal of my work was to investigate, whether it is possible to modify the surface of LRV1 virus-like particles by protein engineering. Next to an LRV1 capsid protein with a C-termini polyhistidine-tag like in the publication, a genetically modified LRV1 capsid protein with an internal polyhistidine-tag was supposed to be expressed. It was supposed to be analyzed, whether it still comes to a self-assembly of the capsid proteins. The genetical constructs were generated by PCR based on a complementary LRV1 DNA, cloned into an expression vector, and using Sanger sequencing it was shown that the amino acid sequences were like expected. For the recombinant expression different E. coli (BL21 STAR (DE3), BL21 (DE3) pLysS, BL21 STAR (DE3) pRARE3 and T7 express lysY/Iq) strains were used, as well as different expression conditions tested by varying temperature, medium, isopropyl-ß-D-thiogalactoside oncentration, and induction duration. Best results were obtained expressing in T7 express lysY/Iq for 5 hours in Terrific Broth medium at 16 °C and 3 mM Isopropyl ß-D-1thiogalactopyranoside. After that, the recombinant proteins were purified under native conditions by their polyhistidine-tags using nickel affinity chromatography. Overall, a higher yield was obtained for the capsid protein with the C-termini polyhistidine-tag during expression, which was also visible in all following experiments. The expression of both constructs in E. coli was comparably low. Still, it was possible to validate for virus-like particles for both proteins using transmission electron microscopy. Thus, it was demonstrated for the first time that surface-modified LRV1 capsid proteins are able to assemble into virus-like particles. For further experiments and higher yield of soluble LRV1 capsid proteins for virus-like particle production, a different expression system should be used: Therefore, the LEXSY expression system should be optimal, it bases on Leishmania tarenolae, a natural LRV1 host system. KW - Protein engineering KW - Recombinant protein expression PY - 2023 SP - 1 EP - 98 PB - Technische Hochschule Wildau CY - Wildau AN - OPUS4-57723 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -