20.500.12556/RUL-122215 Inženiring plaščnega proteina krompirjevega virusa Y za pridobivanje različnih tipov virusom-podobnih delcev. Coat protein engineering of the potato virus y for the production of different types of virus-like particles. Virusom-podobni delci (VLP, angl. virus-like particles) so sestavljeni iz ponavljajočih se enot virusnih strukturnih proteinov, ki se spontano samosestavijo v delce, ki so strukturno zelo podobni ali pa celo identični izvornim virusom. Zaradi odsotnosti virusnega genskega materiala so neinfektivni. Imajo širok nabor uporabe na področju biomedicine ter bionanomaterialov. VLP virusa Y krompirja (PVY, angl. potato virus Y) sestavljajo enote plaščnega proteina (CP, angl. coat protein), povezane v obroče, ki so naloženi vzdolž dolge osi VLP filamenta. Tako tvorijo fleksibilne filamente s premerom 13 nm, ki lahko v dolžino merijo od nekaj 10 nm in vse do 3 µm. CP enoto razdelimo na osrednjo globularno regijo ter iztegnjeni N- in C-končni regiji. N-končna regija je izpostavljena na površini, C-končna regija pa je zakopana v lumen filamenta. Da bi dosegli predstavitev tujih peptidov na zunanji površini filamentov, smo v ekspresijskem sistemu E. coli izražali CP PVY, ki so imeli na N-končni regiji preko genske fuzije vnesena različna peptidna zaporedja. S krajšimi pentapeptidnimi aminokislinskim zaporedji (Asp5, Asn5, Leu5 in His5) smo pokazali, da ko se začetek N-končne regije začne z aminokislinami »MGND«, ki pripadajo originalnemu PVY N-končnemu zaporedju, različna kemijska narava manjšega vključka ne vpliva na samosestavljanje CP v filamente. V primeru fuzije naravnega peptida melitina, dolgega 26 aminokislinskih ostankov, se je protein nahajal v netopni frakciji celičnega lizata. Uspešno pa smo na N-končno regijo vnesli 13 aminokislinskih ostankov dolg SpyTag (ST). S konstruktom ST_CP smo nato preko sistema SpyTag/SpyCatcher pripeli tarčni protein na površino filamenta. Z našimi rezultati smo pokazali, da so VLP PVY primeren sistem za prikaz heterolognih peptidov in proteinov na površini fleksibilnih VLP filamentov. Virus-like particles (VLPs) consist of repeating units of viral structural proteins that spontaneously self-assemble into particles structurally identical or very similar to their native viruses. Due to the absence of viral genetic material they are non-infectious and can be used in a wide range of applications in the field of biomedicine and bionanomaterials. We focused on potato virus Y (PVY) VLPs that form flexible filaments, which can reach up to 3 µm in length and have a diameter of 13 nm. PVY VLPs are assembled from octameric coat protein (CP) rings stacked along the longitudinal axis of filaments. CP unit is comprised of globular core domain and extended N- and C-terminal regions. N-terminal region is located on the surface of the viral filament whereas C-terminal region is buried in the lumen. Using E. coli as an expressions system, we prepared PVY CPs with different peptide sequences introduced in the N-terminal region via genetic fusion, thus achieving the presentation of foreign peptides on the surface of the filaments. With shorter pentapeptide amino acid sequences (Asp5, Asn5, Leu5, and His5), we have shown that by adding “MGND” sequence to the beginning of the N-terminal region, the different chemical nature of the peptide does not affect the self-assembly of CPs. When 26 amino acids residues long melittin was added to the N-terminal region, the protein was present in the insoluble fraction of the cell lysate. But we were successful at introducing 13 amino acids residues long peptide SpyTag (ST) to the N-terminal region of CP. Using SpyTag/SpyCatcher (ST/SC) system we were able to attach the target protein to the surface of the filament composed of ST_CP units. With our results, we have shown that PVY VLPs are a suitable system for displaying heterologous peptides and proteins on the surface of flexible VLP filaments. genetsko inženirstvo virusom-podobni delci virus Y krompirja SpyTag/SpyCatcher genetic engineering virus-like particles potato virus Y SpyTag/SpyCatcher true false false [U. Janjoš] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-11-28 07:15:47 2020-11-28 07:15:56 2022-09-02 04:02:43 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 601.4:577.21:604.6:602.6:579.8(043.2) 187133 43945731 11483.pdf 11483.pdf 1 EFBEF6B11875E66B1EB296133265DC77 b1feb2f5fdd39749f3aaa7c3cf31c708a5319572f2c7c1eff0d9027a6eddf6a3 135ecc31-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=137399 Biotehniška fakulteta 0 0 0