Kloniranje in izražanje proteina NONO v bakterijskih celicah Escherichia coli

Beltram, Ajda (avtor), Župunski, Vera (mentor)

Amiotrofična lateralna skleroza (ALS) je nevrodegenerativna bolezen, ki prizadene predvsem motorične nevrone in povzroči njihovo degradacijo. Odkrili so 20 genov, katerih mutacije naj bi bile povezane z nastankom ALS. Najbolj pogosta mutacija je v genu C9ORF72 (odprti bralni okvir 72 kromosoma 9), kjer pride do povečanja intronskih ponovitev GGGGCC (G4C2)n, kar med drugim povzroči sekvestracijo nekaterih RNA-vezavnih proteinov. Nekatere mutacije pa so v genih, ki zapisujejo proteine parapeg in povzročijo njihovo premestitev iz jedra v citoplazmo, kar vpliva na funkcije parapeg. Parapege so jedrna ribonukleoproteinska telesca brez membrane, sestavljena iz nekodirajoče RNA NEAT1 (jedrni prepis 1 združka parapege) in nanjo vezanih proteinov, izmed katerih sta ključna proteina NONO (oktamer vezavni protein brez domene POU) in SFPQ (spojitveni faktor, bogat s prolini in glutamini). Posledica mutacij, povezanih z ALS, je tudi spremenjeno delovanje proteina NONO, to je RNA- in DNA- vezavni protein, ki spada v družino proteinov DBHS (proteini vedenja Drosophile in človeški spojitveni). Ugotovili so, da se skupaj s proteinom SFPQ veže na prepise ponovitev (G4C2)n, pri čemer nastanejo parapegam podobna telesca, ki so morda eden od patoloških mehanizmov ALS. Da bi lahko preučili interakcije znotraj parapegam podobnih telesc, smo poskusili izraziti protein NONO v fuziji s proteinom, ki veže maltozo (MBP). Pripravili smo dva konstrukta tako, da smo zapis za NONO vstavili v vektor pMCSG7 z zapisom za MBP na N- oziroma C- koncu. Pri izražanju obeh variant v celicah E. coli BL21[DE3] smo fuzijska proteina najprej izolirali z nikljevo afinitetno kromatografijo, pri čemer smo bili v obeh primerih uspešni, a smo varianto z MBP na N-koncu izolirali v večji količini. Poleg tega smo oba fuzijska proteina poskusili izolirati s kolono z afiniteto do MBP, vendar je bil izkoristek manjši kot pri izolaciji z nikljevo afinitetno kromatografijo. Da bi izolirali le protein NONO, smo fuzijskemu proteinu z MBP na N-koncu odcepili oznako His-MBP s proteazo TEV in ga nato očistili s kromatografijo z ločevanjem po velikosti, a smo pri tem imeli velike izgube. Poleg tega pa smo ugotovili, da nobeden od fuzijskih proteinov ni stabilen pri 4 °C, saj sta oba fuzijska proteina agregirala oziroma se razgradila. Izolacijo proteina NONO moramo optimizirati, da bomo dobili ustrezne količine proteina za analizo interakcij v parapegah podobnih telescih.

• Jezik: Slovenski jezik
• Ključne besede: NONO, amiotrofična lateralna skleroza, parapege, izražanje in izolacija proteina
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-139700
• COBISS.SI-ID: 126611971
• Datum objave v RUL: 06.09.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Cloning and expression of protein NONO in Escherichia coli

Izvleček

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons and causes their degradation. They discovered 20 genes whose mutations are thought to be associated with the development of ALS. The most common mutation is in the C9ORF72 gene (chromosome 9 open reading frame 72), where there is an increase in intronic repeats of GGGGCC (G4C2)n, which causes sequestration of some RNA-binding proteins. Some of the mutations are also in the genes that encode paraspeckle proteins and cause their translocation from the nucleus to the cytoplasm, affecting paraspeckle’s functions. Paraspeckles are nuclear ribonucleoprotein bodies without a membrane, consisting of the non-coding RNA NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) and its associated proteins, among which the key proteins are NONO (Non-POU Domain-Containing Octamer-Binding) and SFPQ (Splicing Factor Proline and Glutamine Rich). The mutations associated with ALS also lead to altered function of the NONO protein, an RNA- and DNA-binding protein that belongs to the DBHS (Drosophila Behavior/Human Splicing) protein family. They found that it binds to (G4C2)n-repeat transcripts together with SFPQ protein to form paraspeckle-like bodies, which may be one of the pathological mechanisms of ALS. To investigate the interactions within the paraspeckle-like bodies, we attempted to express the NONO protein in fusion with maltose-binding protein (MBP). We prepared two constructs by inserting the NONO coding sequence into the pMCSG7 vector with the MBP coding sequence at the N- and C- termini. When both variants were expressed in E. coli BL21[DE3] cells, the fusion proteins were first isolated by nickel affinity chromatography, with the variant with MBP at the N- terminus being isolated in higher quantity. We also attempted to isolate both fusion proteins using an MBP affinity column, but this was less efficient than isolation by nickel affinity chromatography. To isolate only the NONO protein, we cleaved the His-MBP tag from the fusion protein with MBP at the N- terminus with TEV protease and then attempted to purify it by size exclusion chromatography, but we had high losses. In addition, we found that neither of the fusion proteins was stable at 4 °C, as both fusion proteins aggregated or degraded. We need to optimize the isolation of the NONO protein to obtain enough of the protein for interaction analysis in paraspeckle-like bodies.

• Ključne besede: NONO, amyotrophic lateral sclerosis, paraspeckles, protein expression and isolation