20.500.12556/RUL-165301 Proizvodnja rekombinantnega s cisteinom bogatega sekrecijskega proteina (CRISP) iz modrasovega strupa in primerjava njegovih biokemijskih lastnosti z naravnim proteinom Production of a recombinant Cys-rich secretory protein (CRISP) from Vipera ammodytes ammodytes venom and comparison of its biochemical properties with the native protein S cisteinom bogati sekrecijski proteini (CRISP) so monomerni proteini z maso 20 do 30 kDa in 10–16 ohranjenimi cisteinskimi ostanki. V tej magistrski nalogi smo želeli pripraviti CRISP-1 iz strupa modrasa (Vipera ammodytes ammodytes) v rekombinantni obliki. Uporabili smo sev »SHuffle K-12« bakterije E. coli z vstavljenim plazmidom pMAL-c5X. VaaCRISP-1 smo izrazili kot fuzijski konstrukt, ki je na svojem N- terminalnem koncu vseboval protein, ki veže maltozo (MBP5), temu je sledilo zaporedje za cepitev z aktiviranim faktorjem X (FXa) in pa zapis za modrasov VaaCRISP-1 (UniProt ID A0A1I9KNM0_VIPAA) brez signalnega zaporedja (rVaaCRISP-1). Po izražanju smo fuzijski protein izolirali s pomočjo afinitetne kromatografije na koloni z amilozno smolo. Cepitev produkta z FXa smo izvedli, ko je bil ta še vezan na koloni ali po eluciji iz nje. Intaktni fuzijski konstrukt je bil stabilen, topen in jasno zaznaven na gelu po gelski elektroforezi. Po cepitvi z FXa pa produkta z maso, ki bi ustrezala rVaaCRISP-1, na gelu ni bilo. Predvidevamo, da zaradi agregacije nepravilno zvitih molekul rVaaCRISP-1 takoj po odstranitvi MBP5. Navkljub temu, da je sev E. coli SHuffle K-12 posebej prilagojen za proizvodnjo proteinov, ki vsebujejo več disulfidnih mostičkov, zvijanje rVaaCRISP-1 ni bilo uspešno. S čiščenjem vzorca na koloni C18 na sistemu HPLC nam je vendarle uspelo izolirati dovolj produkta, da smo z določitvijo njegovega N-terminalnega zaporedja z metodo po Edmanu lahko potrdili, da gre za rVaaCRISP-1. Čeprav smo se trudili optimizirati pogoje izražanja s spreminjanjem koncentracije IPTG, temperature, in časa izražanja, izkoristka nismo uspeli izboljšati nad 10 μg produkta/L kulture. Sklepamo torej, da sev E. coli SHuffle K-12 ni primeren za proizvodnjo rVaaCRISP-1. Cysteine-rich secretory proteins (CRISP) are monomeric proteins with masses of 20 to 30 kDa and 10–16 conserved cysteine residues. In this master's thesis, we aimed to prepare a recombinant form of CRISP-1 from the venom of the nose-horned viper (Vipera ammodytes ammodytes), designated VaaCRISP-1. We used the »SHuffle K-12« strain of E. coli with an inserted pMAL-c5X plasmid. VaaCRISP-1 was expressed as a fusion construct containing the maltose-binding protein (MBP5) at its N-terminal end, followed by a cleavage site for activated factor X (FXa) and the sequence for VaaCRISP-1 (UniProt ID A0A1I9KNM0_VIPAA) sans its signal sequence (rVaaCRISP-1). After expression, the fusion protein was isolated using affinity chromatography on an amylose resin column. Cleavage of the fusion product with FXa was performed while it was still bound to the column or after elution. The intact construct was stable, soluble, and clearly detectable on the gel after gel electrophoresis. However, after cleavage with FXa, no product with a mass corresponding that of rVaaCRISP-1 was visible on the gel. We assume this was due to the aggregation of misfolded rVaaCRISP-1 molecules immediately after the removal of MBP5. Despite the fact that E. coli SHuffle K-12 is specifically desgined for the production of proteins containing multiple disulfide bridges, the folding of rVaaCRISP-1 was unsuccessful. By purifying the sample on a C18 column using a HPLC system, we were nevertheless able to isolate enough product to confirm its N-terminal sequence using Edman degradation, confirming it as rVaaCRISP-1. Although we attempted to optimize the expression conditions by adjusting IPTG concentration, temperature, and expression time, we were unable to improve yields beyond 10 μg of product per liter of culture. Therefore, we conclude that E. coli SHuffle K-12 is not suitable for the production of rVaaCRISP-1. Gadov strup Vipera ammodytes ammodytes CRISP rekombinantni VaaCRISP-1 E. coli bakterijski ekspresijski sistem Viper venom Vipera ammodytes ammodytes CRISP recobinant VaaCRISP-1 E. coli bacterial expression system true false false [V. Golič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-11-30 07:15:43 2024-11-30 07:15:51 2024-12-03 08:03:38 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 577:591.145(043.2) 254768 217369091 45378.pdf 45378.pdf 1 C4272524D69E2C9080FA77422831FEE9 9a8a37ffa5a9860b54494a43527a82b3501fa6224dec21c5bbc2191fcf892687 1f8b4c38-aee2-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=196394 Biotehniška fakulteta 0 0 0