20.500.12556/RUL-161361 Vpliv oznake FLAG na fazno separacijo tekoče-tekoče Impact of FLAG-tagging on liquid-liquid phase separation Fazna separacija tekoče-tekoče (LLPS) je fizikalno-kemijski pojav, pri katerem se raztopina razdeli v dve tekoči fazi, ena z višjo in druga z nižjo koncentracijo topljenca. V diplomskem delu smo raziskovali, kako pri modelnem proteinu nanotelesu 55 (Nb55) prisotnost oznake FLAG z različnim številom ponovitev vpliva na LLPS v odvisnosti od temperature, koncentracije soli, koncentracije proteina in koncentracije polietilen glikola. V ta namen smo z uporabo kloniranja s sestavljanjem in vivo pripravili DNA konstrukte, ki so nosili zapis za nanotelo z 0x (brez oznake), 1x, 2x in 3x oznako FLAG. Pojav LLPS smo spremljali z UV-Vis spektroskopijo ob hlajenju s hitrostjo 0,1 °C/min. Ugotovili smo, da do pojava LLPS pride pri temperaturah med 15 do 5 °C, če ima protein zadostno število ponovitev oznake FLAG. Potrdili smo povezavo med koncentracijo proteina in med nastankom LLPS, saj je do pojava pri vzorcih z višjimi koncentracijami prišlo že pri višjih temperaturah (okoli 15 °C), kot pri tistih z nižjimi koncentracijami (okoli 5 °C). Potrdili smo tudi zvezo med številom ponovitev oznake FLAG in nagnjenostjo k nastanku LLPS, saj sta proteina z 2x in 3x oznako FLAG edina pri katerih smo zaznali LLPS v preiskovanem koncentracijskem območju. Dodatek soli ali polietilen glikola na pojav LLPS deluje zaviralno. Liquid-liquid phase separation (LLPS) is a physico-chemical phenomenon in which a solution spontaneously separates into two liquid phases, with one having a higher and one having a lower concentration of solute. Our research focused on how temperature, salt concentration, protein concentration, addition of crowding agents (eg. polyethylene glycol) and addition of a variable number of FLAG-tag repeats affects LLPS apparition of model protein nanobody 55 (Nb55). For this purpose, we used in vivo assembly cloning to create DNA constructs encoding nanobodies with 0x (without FLAG-tag), 1x, 2x and 3x repeats of the FLAG-tag. We studied the phenomenon using UV-Vis spectroscopy while cooling our samples at a rate of 0,1 °C/min. We discovered that LLPS manifests at temperatures ranging between 15 and 5 °C, if the protein has an adequate number of FLAG repeats. We confirmed a link between protein concentration and LLPS apparition, as samples with higher protein concentrations underwent LLPS at higher temperatures (around 15 °C), than those with lower protein concentrations (around 5 °C). We also established a link between the number of FLAG-tag repeats and the protein's propensity to undergo LLPS, as the proteins with 2x and 3x FLAG-tag repeats are the only ones which underwent LLPS in our studied concentration range. The addition of salt or polyethylene glycol inhibitis LLPS apparition. fazna separacija tekoče-tekoče oznaka FLAG FPLC UV-Vis IVA liquid-liquid phase separation FLAG-tag FPLC UV-Vis IVA true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2024-09-10 10:45:00 2024-09-10 10:45:10 2024-09-12 03:25:05 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 24114 206978051 Koren_Ziga_-_Vpliv_oznake_FLAG_na_fazno_separacijo_tekoce-tekoce.pdf Koren_Ziga_-_Vpliv_oznake_FLAG_na_fazno_separacijo_tekoce-tekoce.pdf 1 B76F4A0B4565A56CAA3858E5D6590861 c93102c4f8189163ae0b837fe7882460c5956d086c4e99a01f30f080c9e385e7 f3501e01-6f50-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=190278 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0