diplomsko delo
Ana Pervanja (Author), Vera Župunski (Mentor)

Abstract

Spojitveni faktor, bogat s prolini in glutamini (SFPQ), je protein, ki lahko interagira tako s proteini kot tudi z RNA in DNA. Posledično sodeluje pri mnogih procesih v celici in ima vse večji pomen v raziskavah, ki so povezane z nevrodegenerativnimi boleznimi. Cilj diplomske naloge je bil izraziti in izolirati protein SFPQ v taki količini in čistosti, ki bi omogočila nadaljnje eksperimente, npr. spremljanje nastanka parapeg in vitro. Pripravili smo vektorja pMCSG7-MBP-SFPQ (N-končna fuzija z MBP in His6) in pMCSG7-SFPQ (SFPQ brez fuzij). SFPQ smo nato izražali z izhodiščnim vektorjem pJ4M-SFPQ-MBP in z vektorjem pMCSG7-MBP-SFPQ. Fuzija s His6 je omogočila izolacijo proteina s pomočjo afinitetne kromatografije z imobiliziranimi kovinskimi ioni (IMAC). Konstrukt SFPQ-MBP se je razgradil že med izražanjem. MBP-SFPQ se je izražal v dovolj velikih količinah, a je pri postopkih izolacije prišlo do razgradnje. Z vektorjem pMCSG7-SFPQ smo izražali SFPQ brez fuzij in ga izolirali z dodatkom cinkovega klorida. Do proteolize je prišlo pri izražanju in/ali izolaciji. Sklepali smo, da je vzrok za razgradnjo proteina velik delež neurejene strukture, ki je zato bolj občutljiv na proteaze. Postopke bi morali optimizirati in zmanjšati aktivnost proteaz.

Keywords

spojitveni faktor, bogat s prolini in glutamini;protein SFPQ;izražanje;amiotrofična lateralna skleroza;ALS;frontotemporalna demenca;FTD;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Pervanja]
UDC: 577.21:577.112(043.2)
COBISS: 164612355 Link will open in a new window
Views: 16
Downloads: 3
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Other data

Secondary language: English
Secondary title: Cloning and expression of the protein SFPQ
Secondary abstract: Splicing factor proline and glutamine rich (SFPQ) is a protein that interacts with protein, RNA and DNA. Because of that, it has a plethora of cell functions and is increasingly popular among neurodegenerative disease studies. Our aim was to express and purify enough SFPQ to conduct experiments such as monitoring the formation of paraspeckles. Vectors pMCSG7-MBP-SFPQ (N-terminal fusion with MBP and His6) and pMCSG7- SFPQ (untagged SFPQ) were prepared. SFPQ was then expressed using pJ4M-SFPQMBP (prepared in the lab) and pMCSG7-MBP-SFPQ vectors. The fusion with His6 allowed the protein to be purified by immobilized metal ion affinity chromatography (IMAC). SFPQ-MBP was already degraded during expressing. MBP-SFPQ was expressed in sufficient amounts, but degradation occurred during the purification steps. We used the pMCSG7-SFPQ vector to express untagged SFPQ and isolate it by adding zinc chloride. Proteolysis occurred upon expression and/or purification. We concluded that the cause of protein degradation is a large proportion of the disordered structure, which is more sensitive to proteases. The procedures should therefore be optimized to reduce the activity of proteases.
Secondary keywords: SEPQ;expressing;ALS;FTD;Beljakovine;Nevrodegenerativne bolezni;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: 38 str.
ID: 19909001