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Priprava rekombinantnega aneksina A11 v bakterijah E. coli z uporabo ekspresijskih vektorjev na osnovi pMCSG7
Maklin, Ana (Author), Župunski, Vera (Mentor) More about this mentor... This link opens in a new window

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Abstract
Amiotrofična lateralna skleroza (ALS) je progresivna in smrtna bolezen, ki jo povzroči degeneracija motoričnih nevronov. Ne dolgo nazaj so z ALS povezali mutacije v genu ANXA11. Aneksin A11 (ANXA11) spada v naddružino aneksinov, strukturno sorodnih proteinov, ki se v prisotnosti kalcija vežejo na celično membrano. Večino proteinske strukture sestavljajo štiri visoko ohranjena homologna α-vijačna zvitja, medtem ko je N-končna regija zelo variabilna. N-konec ANXA11 je nestrukturiran in najdaljši v družini aneksinov. Pomemben je za jedrno lokalizacijo in razgradnjo ANXA11. Rekombinantni protein je v svojem magistrskem delu že pripravil in izoliral študent Jakob Rupert, pri čemer je uporabil vektorja pET28a in pET14b. Zaradi težav pri izolaciji smo se odločili za drug vektorski sistem. Z metodo od ligacije neodvisnega kloniranja LIC smo zapis za ANXA11 wt vstavili v bakterijska ekspresijska vektorja pMCSG7 in pMCSG7-GST. Zanimalo nas je, če lahko fuzija rekombinantnega ANXA11 z oznako GST zmanjša fragmentacijo v procesu izražanja in izolacije proteina. Po uspešnem testnem izražanju ANXA11 v bakterijskih celicah E. coli BL21[DE3] pLysS smo nadaljevali s proizvodnjo v večjem merilu. Proteine smo izolirali z Ni2+- ali glutation-S-transferazno afinitetno kromatografijo. ANXA11 brez oznake GST se je delno fragmentiral, verjetno zaradi nestrukturiranega N-končnega dela, ANXA11 z GST pa se je izkazal kot bolj stabilen. Proteinu ANXA11-GST smo uspešno odcepili oznako His-GST s proteazo TEV. Rekombinante proteine smo dokončno kromatografsko očistili z ločevanjem po velikosti.

Language:Slovenian
Keywords:aneksin A11, amiotrofična lateralna skleroza, proteinski agregati, izolacija proteina
Work type:Bachelor thesis/paper (mb11)
Tipology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2019
COBISS.SI-ID:1538397379 This link opens in a new window
Views:754
Downloads:250
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Secondary language

Language:English
Title:Preparation of recombinant annexin A11 in E. coli using pMCSG7-based expression vectors
Abstract:
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease caused by degeneration of motor neurons. Not until recently ALS has been associated with specific mutations in ANXA11 gene. Annexin A11 (ANXA11) belongs to a larger annexin family of structurally related Ca2+-dependent-membrane-binding proteins. The C-terminal core contains four highly conserved homologous alpha helical repeats. Contrary, the N-terminus is highly variable, structurally disordered and the longest in the annexin family. It is important for nuclear localization and degradation of ANXA11. Recombinant ANXA11 was previously prepared by master's student Jakob Rupert, using pET28a and pET14b vectors. Due to protein degradation problems we decided to attempt production using an alternative vector system. With ligation independent cloning we incorporated ANXA11 sequence in bacterial expression vectors pMCSG7 and pMCSG7-GST. We wanted to know, if the fusion of recombinant protein with GST-tag could reduce fragmentation in the process of protein expression and isolation. After successful test expression of ANXA11 in E. coli strain BL21[DE3] pLysS, we continued with ANXA11 production on a higher scale. Proteins were isolated using Ni2+- or glutathione-S-transferase affinity chromatography. While ANXA11 without GST tended to show some fragmentation, probably due to its flexible N-terminal tail, ANXA11 with GST seemed to be more stable. We successfully cleaved His-GST tag from ANXA11-GST protein with protease TEV. We finally purified recombinant ANXA11 with size-exclusion chromatography.

Keywords:annexin A11, amyotrophic lateral sclerosis, protein aggregation, protein isolation

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