diplomsko delo
Klara Razboršek (Author), Boris Rogelj (Mentor)

Abstract

Parapege so nemembranski organeli, ki se nahajajo znotraj jedra. Sestavljene so iz dolge nekodirajoče RNA (lncRNA) in različnih RNA-vezavnih proteinov, kot sta izrezovalni faktor bogat s prolini in glutamini (SFPQ) in protein brez domene POU, ki se veže na oktamer (NONO). Osrednji del tvori lncRNA NEAT1 (jedrni prepis 1 združka parapege). Heterogeni jedrni ribonukleoprotein K (hnRNPK) je RNA-vezavni protein, ki se nahaja v jedru in je eden ključnih proteinov pri nastanku parapeg. Povečana prisotnost teh jedrnih organelov v nevronih je značilna za amiotrofično lateralno sklerozo (ALS). Vloga in nastanek parapeg v povezavi z ALS še ni poznana. Za študij parapeg v povezavi z ALS smo se v sklopu diplomskega dela osredotočili na pripravo proteina parapeg hnRNPK. V ta namen smo s sestavljanjanjem po Gibsonu zapisa za HNRNPK in HNRNPK-NeonGreen vstavili v bakterijski ekspresijski vektor pMCSG7. Oba konstrukta HNRNPK smo uspešno poskusno izrazili v bakterijskih celicah E. coli BL21[DE3]. Določili smo optimalne pogoje za izražanje, ki se bodo uporabili pri obsežnejši proizvodnji proteina HNRNPK. Za nadaljnje raziskave je potrebno proces izražanja izvesti v večjem obsegu in izvesti še postopke čiščenja posameznih proteinov.

Keywords

parapege;amiotrofična lateralna skleroza;ALS;heterogeni jedrni ribonukleoprotein K;protein HNRNPK;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: [K. Razboršek]
UDC: 577.112:602.7(043.2)
COBISS: 167167747 Link will open in a new window
Views: 56
Downloads: 21
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Other data

Secondary language: English
Secondary title: Molecular cloning and preparation of the paraspeckle protein HNRNPK in Escherichia coli
Secondary abstract: Paraspeckles are nuclear membraneless organelles. They consist of long non-coding RNA (lncRNA) and various RNA-binding proteins such as splicing factor proline and glutamine rich (SFPQ) and Non-POU domain containing octamer binding protein (NONO). They form around the lncRNA NEAT1 (nuclear paraspeckle assembly transcript 1). Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein located in the nucleus and is one of the key proteins in paraspeckle formation. An increased abundance of these nuclear organelles in neurons is characteristic of amyotrophic lateral sclerosis (ALS). The role and formation of paraspeckles in the context of ALS is not yet known. In this work, we focused on the preparation of the paraspeckle protein hnRNPK to study paraspeckles in association with ALS. To this end, we incorporated the HNRNPK and HNRNPK-NeonGreen sequences into a bacterial expression vector pMCSG7 using Gibson assembly. We successfully expressed both HNRNPK constructs in the E. coli strain BL21[DE3] and determined the optimal conditions for expression that can be used for larger-scale production of HNRNPK. For further research, it is necessary to carry out the expression process on a larger scale and carry out purification procedures for individual protein constructs.
Secondary keywords: paraspeckles;HNRNPK;ALS;molecular cloning;protein expression;Beljakovine;Molekularno kloniranje;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: 50 str.
ID: 19892386