20.500.12556/RUL-184540 Kloniranje in izolacija ORF1p iz LINE1 z vezavnim proteinom za maltozo Cloning and isolation of LINE1 ORF1p with maltose-binding protein Protein ORF1p iz retrotranspozona LINE1, edinega aktivnega avtonomnega retrotranspozona v človeškem genomu, je približno 40 kDa velik RNA-vezavni protein z aktivnostjo šaperona nukleinskih kislin. V zadnjih letih ima vse večji pomen v raziskavah, ki so povezane z nevrodegenerativnimi boleznimi. Cilj diplomske naloge je bil izraziti in izolirati protein ORF1p v zadostni količini za nadaljnje eksperimente, kot je test jedrnega uvoza. Pripravili smo vektorja pMCSG7-MBP-ORF1p (N-končna fuzija z MBP in His6) za izražanje zapisa za MBP-ORF1p ter pMCSG7-ORF1p-MBP (C-končna fuzija z MBP in His6) za izražanje zapisa za ORF1p-MBP. Oznaka His6 je omogočila izolacijo proteina z afinitetno kromatografijo na imobiliziranih kovinskih ionih (IMAC), medtem ko je MBP omogočil izolacijo z MBP-afinitetno kromatografijo na dekstrin-sefarozni koloni. Konstrukt ORF1p-MBP se je med izražanjem in izolacijo razgradil v večji meri kot MBP-ORF1p. MBP-ORF1p smo z obema metodama izolirali s primerljivo čistostjo. V obeh primerih je bilo v elucijskih frakcijah prisotnih veliko razgradnih produktov MBP-ORF1p, kar smo potrdili z imunodetekcijo. Sklepali smo, da je vzrok razgradnje proteina prisotnost intrinzično neurejenih regij na ORF1p, ki so bolj dovzetne za proteolizo. Postopek izolacije smo delno izboljšali z dodatkom glicerola v vezavne in elucijske pufre. Postopke bi morali dodatno optimizirati in zmanjšati aktivnost proteaz. Oznako His6-MBP smo z MBP-ORF1p uspešno odstranili s proteazo TEV. The ORF1p protein encoded by the LINE1 retrotransposon, the only active autonomous retrotransposon in the human genome, is an approximately 40 kDa RNA-binding protein with nucleic acid chaperone activity. In recent years, it has gained increasing attention in research related to neurodegenerative diseases. Our aim was to express and isolate ORF1p in sufficient quantity for further experiments, such as a nuclear import assay. We constructed the vectors pMCSG7-MBP-ORF1p (N-terminal fusion with MBP and His6) for the expression of MBP-ORF1p and pMCSG7-ORF1p-MBP (C-terminal fusion with MBP and His6) for the expression of ORF1p-MBP. The His6 tag enabled protein purification by immobilized metal affinity chromatograph (IMAC), while the MBP tag allowed purification by MBP affinity chromatography on a dextrin-sepharose column. The ORF1p-MBP construct exhibited a higher degree of degradation during expression and purification compared to MBP-ORF1p. MBP-ORF1p was isolated with comparable purity using both purification methods. In both cases, a substantial amount of degradation products was detected in the elution fractions, as confirmed by immunodetection. We concluded that protein degradation was likely due to the presence of intrinsically disordered regions in ORF1p, which are more susceptible to proteolysis. The isolation procedure was partially improved by the addition of glycerol to the binding and elution buffers. Further optimization of the protocol and reduction of protease activity would be necessary. The His6-MBP tag was successfully removed from MBP-ORF1p using TEV protease. ORF1p LINE1 izolacija ALS FTD ORF1p LINE1 isolation ALS FTD true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-07-09 13:10:03 2026-07-09 13:10:15 2026-08-20 13:09:14 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25574 288335875 Kramar_Zala_-_Kloniranje_in_izolacija_ORF1p_iz_LINE1_z_vezavnim_proteinom_za_maltozo.pdf Kramar_Zala_-_Kloniranje_in_izolacija_ORF1p_iz_LINE1_z_vezavnim_proteinom_za_maltozo.pdf 1 76F1810E34EF2423D0A94A8F3C7D6588 cec1286b7ea2657aef172c4193a11c302ea0ab627c6dacaf5c230d077b4913c0 a4ff2ba8-7b86-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237428 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0