20.500.12556/RUL-171704 Vpliv proregije na stabilnost in aktivnost rekombinantnega pernizina The impact of proregion on stability and activity recombinant pernisine Pernizin je subtilizinu podobna alkalna endopeptidaza iz hipertermofilne arheje Aeropyrum pernix, znana po izjemni termostabilnosti in zmožnosti razgradnje prionov. Podobno kot bakterijski subtilizin se tudi pernizin v arheji sintetizira kot neaktivni prekurzor in potrebuje pomoč N-terminalne pro-sekvence (proregije) za zvitje v zreli encim. Ta pri subtilizinu deluje ne le kot inhibitor, ki pred odcepom inhibira aktivno mesto encima, temveč tudi kot molekularni šaperon, ki omogoča pravilno zvijanje katalitične domene. Zvitje pernizina pa za razliko od subtilizina, katerega zvitje je povsem odvisno od šaperonskega delovanja proregije, omogočijo ioni Ca2+, ki se vežejo v osem mest na katalitični domeni. Že vzpostavljeni postopki sinteze rekombinantnega pernizina v bakterijah Escherichia coli in Streptomyces rimosus brez proregije so pokazali, da ta ohrani visoko termostabilnost in aktivnost za preproste substrate. V diplomski nalogi smo preverili, ali poleg ionov Ca2+ tudi proregija v določeni meri vpliva na konformacijo, stabilnost in aktivnost katalitične domene pernizina. Uspešno smo sintetizirali pernizin s proregijo v bakterijah E. coli, medtem ko sinteza pernizina brez proregije ni bila uspešna. Zato smo se osredotočili na in vitro zvijanje aktiviranega pernizina z in brez proregije po predhodni denaturaciji. Z uporabo različnih metod, kot so diferenčna dinamična fluorimetrija, spektroskopija s cirkularnim dikroizmom, fluorescenčna emisijska spektrometrija in razgradnja kompleksnega substrata, smo analizirali konformacijo, stabilnost in proteolitično aktivnost obeh in vitro zvitih različic. Proregija ni pokazala pomembnega vpliva pri nobeni od analiz, torej ne vpliva na nobeno od navedenih lastnosti. Rezultati analiz potrjujejo prejšnja predvidevanja, da proregija deluje le kot inhibitor, ne pa tudi kot šaperon za katalitično domeno. Pernisine is a subtilisin-like alkaline endopeptidase from the hyperthermophilic archaeon Aeropyrum pernix, known for its exceptional thermostability and ability to degrade prions. Like bacterial subtilisin, pernisine is synthesized in the archaeon as an inactive precursor and requires the assistance of an N-terminal pro-sequence (proregion) for folding into the mature enzyme. In subtilisin, the proregion functions not only as an inhibitor that blocks the active site prior to cleavage, but also as a molecular chaperone that enables correct folding of the catalytic domain. In contrast, the folding of pernisine is facilitated by Ca²⁺ ions, which bind to eight sites on the catalytic domain, whereas subtilisin folding is entirely dependent on the chaperone-like function of the proregion. Established methods for recombinant synthesis of pernisine in Escherichia coli and Streptomyces rimosus without the proregion have shown that the enzyme retains high thermostability and activity towards simple substrates. In this thesis, we investigated whether, in addition to Ca²⁺ ions, the proregion also influences the conformation, stability, and activity of the catalytic domain of pernisine. We successfully synthesized pernisine with the proregion in E. coli, whereas synthesis without the proregion was unsuccessful. Therefore, we focused on in vitro refolding of activated pernisine with and without the proregion after prior denaturation. Using various methods such as differential scanning fluorimetry, circular dichroism spectroscopy, fluorescence emission spectrometry, and degradation of a complex substrate, we analyzed the conformation, stability, and proteolytic activity of both in vitro refolded variants. The proregion did not show a significant influence in any of the analyses, indicating it does not affect any of the aforementioned properties. The results confirm previous assumptions that the proregion functions solely as an inhibitor and not as a chaperone for the catalytic domain. Pernizin proteaza rekombinantni protein proregija proteolitična aktivnost termostabilnost Pernisine protease recombinant protein proregion proteolytic activity thermostability true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2025-08-31 07:16:06 2025-08-31 07:16:13 2025-09-15 03:55:32 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 261882 247497475 50741.pdf 50741.pdf 1 62953C26C0C0029AB35EAE83DF58BB62 aacbc1890d47fa7cb7f1efb85d1dcbc026313128316ab32664c1497be069d7c5 79011e8f-8629-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215086 Biotehniška fakulteta 0 0 0