20.500.12556/RUL-190924 Kloniranje in izražanje človeške kaspaze-1 ter vrednotenje njene encimske aktivnosti Cloning and expression of human caspase-1 and its enzymatic activity evaluation Kaspaza-1 je cisteinsko-aspartatna proteaza, ki ima ključno vlogo pri vnetnem odzivu, saj omogoča zorenje provnetnih citokinov interlevkina-1β in interlevkina-18 ter sodeluje pri piroptozi. Zaradi svoje pomembne vloge pri razvoju številnih bolezni predstavlja pomembno tarčo za razvoj novih zdravilnih učinkovin. Namen magistrskega dela je bil pripraviti rekombinantno človeško kaspazo-1 za uporabo pri vrednotenju potencialnih selektivnih zaviralcev, sintetiziranih na Katedri za farmacevtsko kemijo Fakultete za farmacijo Univerze v Ljubljani, ter testirati nekatere izmed njih. Pripravili smo dva ekspresijska plazmida, prvi je vseboval zapis za podenoto p10, drugi zapis za podenoto p20 človeške kaspaze-1. Izhajali smo iz komercialne komplementarne DNA človeške kaspaze-1, iz katere smo pomnožili zapisa za obe podenoti. Po uspešni ligaciji s plazmidom pET22b smo z vektorjema pET22b/p10 in pET22b/p20 transformirali klonirni sev E. coli TOP10. Konstrukta smo potrdili s sekvenciranjem po Sangerju ter z njima nadalje transformirali ekspresijski sev E. coli BL21-CodonPlus. Izvedli smo tri poskuse ekspresije in izolacije rekombinantne kaspaze-1 po dveh protokolih. Pri prvem poskusu po čiščenju proteina s preparativnim kromatografskim sistemom ÄKTA prisotnosti proteina v izbranih frakcijah s specifičnimi protitelesi z metodo prenosa western nismo potrdili. Pri drugem in tretjem poskusu kljub različnim pristopom k optimizaciji postopka zaradi težav z obarjanjem proteina nismo uspeli pridobiti topne in encimsko aktivne kaspaze-1. Zaradi neuspešne izolacije smo za vrednotenje zaviralcev uporabili komercialno dostopen rekombinantni encim. Referenčni zaviralec NSC697923 je izkazal zaviralno delovanje v nanomolarnem območju, medtem ko je spojina KEM-31A, sintetizirana na Fakulteti za farmacijo, zavirala kaspazo-1 v nizkem mikromolarnem območju. Spojina KEM-31A izkazuje obetavno zaviralno delovanje in predstavlja primerno izhodiščno spojino za nadaljnjo optimizacijo potencialnih zaviralcev kaspaze-1. Poleg tega smo pripravili osnovo za nadaljnjo optimizacijo postopka priprave rekombinantne človeške kaspaze-1. Caspase-1 is a cysteine-aspartate protease that plays a key role in the inflammatory response by enabling the maturation of the pro-inflammatory cytokines interleukin-1β and interleukin-18 and participating in pyroptosis. Due to its important role in the development of numerous diseases, caspase-1 represents an important target for the development of new active pharmaceutical ingredients. The aim of this master’s thesis was to prepare recombinant human caspase-1 for evaluating potential selective inhibitors synthesized at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana. Two expression plasmids were prepared: the first contained the coding sequence for the p10 subunit of human caspase-1, while the second contained the coding sequence for the p20 subunit. Commercially available human caspase-1 complementary DNA was used as the starting material, from which the coding sequences for both subunits were amplified. Following successful ligation into the pET22b plasmid, the pET22b/p10 and pET22b/p20 vectors were used for transformation of the cloning strain E. coli TOP10. The constructs were confirmed by Sanger sequencing and subsequently used for transformation of the expression strain E. coli BL21-CodonPlus. Three experiments for the expression and isolation of recombinant caspase-1 were performed using two protocols. In the first experiment, following protein purification using the ÄKTA preparative chromatography system, the presence of the protein in the selected fractions was not confirmed by western blot analysis using specific antibodies. In the second and third experiments, despite various approaches to process optimization, soluble and enzymatically active caspase-1 could not be obtained due to difficulties with protein aggregation. Because the isolation was unsuccessful, a commercially available recombinant enzyme was used to evaluate the inhibitors. The reference inhibitor NSC697923 exhibited inhibitory activity in the nanomolar range, whereas the compound KEM-31A, synthesized at the Faculty of Pharmacy, inhibited caspase-1 in the low micromolar range. KEM-31A demonstrated promising inhibitory activity and represents a suitable lead compound for further optimization of potential caspase-1 inhibitors. In addition, the groundwork was established for further optimization of the recombinant human caspase-1 preparation process. kaspaza-1 rekombinantni protein izražanje v E. coli inkluzijska telesca zaviralci kaspaze-1 caspase-1 recombinant protein expression in E. coli inclusion bodies caspase-1 inhibitors true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-11-07 08:45:07 2026-11-07 08:45:22 2026-11-08 04:05:45 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 138523 40577.pdf 40577.pdf 1 4AB19EDC99274230F8B46AB53BA329EC 2c20694aba13a27957aa5c68bacb10c2b9176e47119dca7ecb647b9fd80422ae 1a077890-da7f-11f1-aea2-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=252726 Fakulteta za farmacijo 0 0 0