diplomsko delo
Špela Rapuš (Author), Boris Rogelj (Mentor)

Abstract

Razširitvena mutacija G4C2 na genu c9orf72 po več znanih mehanizmih vpliva na razvoj nevrodegenerativnih bolezni kot sta ALS in FTD. Eden izmed njih je tudi pridobitev toksičnih lastnosti zaradi smernih in protismernih prepisov RNA. V primeru vezave protismernega prepisa RNA na protein fenilalanin tRNA-sintetazo (FARS) pride do inhibicije aminoacilacije tRNAPhe, kar negativno vpliva na potek bolezni. Da bi bolje raziskali način vezave in interakcije protismernega prepisa RNA s proteinom FARS, smo protein izrazili v bakterijskem ekspresijskem sistemu in ga izolirali. Postopek izražanja smo optimizirali tako, da smo večino proteina dobili v topni frakciji za kar smo uporabili maltoza vezavni protein. Obe podenoti proteina FARS sta se najbolje izražali pri 18 ⁰C z dodanim fuzijskim maltoza vezavnim proteinom. Proteina smo izolirali z nikljevo afinitetno kromatografijo z uporabo heksahistidinske oznake na obeh rekombinantnih proteinih. V prihodnosti bi morali izvesti izražanje in izolacijo v večjem merilu, postopek izolacije pa bi bilo potrebno tudi optimizirati, saj je bil velik del rekombinantnih proteinov proteolitično razgrajen. Boljše poznavanje mehanizma bi lahko prispevalo k razvoju novih metod zdravljenja bolezni, povezanih s c9orf72.

Keywords

amiotrofična lateralna skleroza;ALS;frontotemporalna demenca;FTD;fenilalanin-tRNA sintetaza;FARS;gen c9irf72;izražanje proteinov;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [Š. Rapuš]
UDC: 577.21:577.112(043.2)
COBISS: 177421827 Link will open in a new window
Views: 43
Downloads: 7
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Other data

Secondary language: English
Secondary title: Expression and Isolation of the Phenylalanyl-tRNA Synthetase Protein
Secondary abstract: The G4C2 expansion mutation in the c9orf72 gene affects the development of neurodegenerative diseases such as ALS and FTD by several known mechanisms. One of them is the acquisition of toxic functions due to sense and antisense RNA transcripts. In the case of binding of the antisense RNA transcript to the protein phenylalanine tRNA- synthetase, inhibition of aminoacylation of tRNAPhe occurs, which has a negative impact on the course of the disease. To investigate the mode of binding and interaction, we have expressed and isolated protein using a bacterial expression system. The expression process was optimized to obtain as much protein as possible in the soluble fraction. For that purpose, we used maltose binding protein. Both subunits of the FARS protein were best expressed at 18 ⁰C with added fusion partner maltose binding protein. Proteins were isolated by nickel affinity chromatography using hexahistidine tags on both recombinant proteins. Future studies of expression and isolation should be performed on a larger scale. The isolation procedure would also have to be optimized since a lot of proteolytic degradation was detected in the present work. A better understanding of the mechanism could contribute to the development of new treatments for c9orf72-related diseases.
Secondary keywords: ALS;FTD;FARS;c9org72 gene;protein expression;Beljakovine;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 45 str.
ID: 21372105