diplomsko delo
Povzetek
Prepisana heksanukleotidna razširitvena mutacija gena C9orf72 predstavlja osnovo za
izgradnjo toksičnih nemembranskih skupkov G4C2. Te se pogosto pojavljajo pri
družinskih različicah amiotrofične lateralne skleroze in frontotemporalne demence.
Mehanizmi nastanka toksičnih skupkov niso dobro poznani in bi lahko bili temelj novega
načina zdravljenja bolezni. Za raziskave skupkov so potrebni proteini, ki se kolokalizirajo
z mutiranim prepisom in tvorijo skupke G4C2. Zato smo v okviru diplomske naloge
pripravili plazmide za in vitro transkripcijo/translacijo in za izražanje proteinov SFPQ,
NONO, PABPC1, TDP-43 ter FUS v bakterijah E. coli BL21(DE3). Nemutiranim
različicam proteinov smo dodali fuzijski maltoza vezavni protein za povečano topnost
konstruktov in fluorescenčni protein NeonGreen. Plazmid je vseboval tudi
heksahistidinsko oznako in cepitveno mesto TEV za lažjo izolacijo konstruktov. Proteine
SFPQ, PABPC1, TDP-43 in FUS smo izražali pri 37 oC, 1 mM IPTG, tri ure in pri 30 oC,
0,5 mM IPTG, pet ur. Najboljši pogoji za izražanje proteina so bili pri nižji temperaturi
in nižji koncentraciji induktorja, ker se je takrat večina proteina nahajala v topni frakciji.
V prenizkih količinah za nadaljnjo izolacijo sta se izražala konstrukta SFPQ in SFPQNeonGreen. V nadaljevanju je potrebno preveriti izražanje konstruktov NONO, NONONeonGreen in NeonGreen-PABPC1 ter z uporabo Ni2+ ionov izolirati izražene proteine,
da dosežemo naš cilj.
Ključne besede
amiotrofična lateralna skleroza;ALS;frontotemporalna demenca;FTD;nemembranski organeli;izražanje proteinov;gen C9orf72;skupki G4C2;SFPQ;TDP-43;NONO;PABPC1;FUS;diplomska dela;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2021 |
Tipologija: |
2.11 - Diplomsko delo |
Organizacija: |
UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo |
Založnik: |
[T. Nograšek] |
UDK: |
577.21:577.112(043.2) |
COBISS: |
83269635
|
Št. ogledov: |
307 |
Št. prenosov: |
59 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Cloning and optimization of expression of recombinant proteins of Neat1 and G4C2 clusters for in vitro observation of their formation |
Sekundarni povzetek: |
C9orf72 hexanucleotide repeat expansion transcript is the foundation on which toxic
membraneless G4C2 foci are built. These are common among patients with familial
amyotrophic lateral sclerosis and frontotemporal dementia. Mechanisms underlying the
formation of toxic G4C2 foci are unknown and could represent the basis for new
treatments. To research G4C2 foci proteins that localize and form these structures are
needed. For that reason, this work focuses on the production of plasmids for in vitro
transcription/translation and expression of SFPQ, NONO, PABPC1, TDP-43 and FUS
proteins in E. coli BL21(DE3) bacteria. A fluorescent NeonGreen protein was fused to
the wild type proteins along with maltose binding protein for increased construct
solubility. The plasmids also contained a hexahistidine tag and a TEV cleavage site to
assist with protein purification. SFPQ, PABPC1, TDP-43 and FUS were expressed at 37
oC with 1 mM IPTG for three hours and at 30 oC, with 0,5 mM IPTG for five hours.
Lower temperature expression yielded a better result since most of the protein was in the
soluble fraction of the cell lysate. Both SFPQ and SFPQ-NeonGreen were expressed in
too low a quantity for further protein isolation. Further work needs to check the expression
of NONO, NONO-NeonGreen, NeonGreen-PABPC1 and purify all of the constructs to
achieve our goal. |
Sekundarne ključne besede: |
membraneless organelles;protein expression;G4C2 foci; |
Vrsta dela (COBISS): |
Diplomsko delo/naloga |
Študijski program: |
1000371 |
Konec prepovedi (OpenAIRE): |
1970-01-01 |
Komentar na gradivo: |
Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija |
Strani: |
IX, 59 str. |
ID: |
13381031 |