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Izolacija in karakterizacija mutantov aneksina A11 D40G in ΔN1-118
Supej, Špela (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 neozdravljiva nevrodegenerativna bolezen, ki jo povzroči progresivna degeneracija motoričnih nevronov v možganski skorji, možganskem deblu in hrbtenjači. Bolezen so do sedaj povezali z mutacijami več kot 100 genov, med drugimi tudi z mutacijami na genu ANXA11. Aneksin A11 (ANXA11) je 505 aminokislinskih ostankov dolg protein, ki spada v naddružino aneksinov in se v odvisnosti od kalcija veže na fosfolipidne membrane. Protein se deli na dva strukturno povsem različna dela. Na C-koncu vsebuje štiri aneksinske ponovitve, sestavljene iz petih α-vijačnic, N-končna regija pa je v primerjavi s C-končno izredno variabilna in neurejena. Struktura N-končnega repa zaradi svojih intrinzičnih lastnosti še ni bila določena. V okviru diplomskega dela smo želeli pridobiti večje količine skrajšane oblike proteina, ki je imel N-končno domeno skrajšano za prvih 118 aminokislin do Ser119 (označili smo ga ANXA11dN1-118), in ANXA11dN-ter, ki je bil skrajšan do Gly179. Zapisa za rekombinantni obliki proteina smo z metodo od ligacije neodvisnega kloniranja LIC vnesli v bakterijska ekspresijska vektorja pMCSG7 in pMCSG7-GST. Zanimalo nas je, ali bo oznaka GST pomagala pri povečanju topnosti proteina in zmanjšala njegovo cepitev, ki se je pojavila v predhodnih poskusih z drugimi vektoji. Obe obliki smo izrazili v bakterijskih celicah E. coli seva BL21 [DE3] pLysS in ju izolirali s postopkom nikljeve afinitetne kromatografije. Uspelo nam je pridobiti 19,2 mg proteina ANXA11dN1-118 in 6,4 mg ANXA11dN-ter, kar je zadostovalo za nadaljnje kristalizacijske poskuse. Med izolacijo ni prihajalo do cepitve proteinov. Prav tako smo v protein ANXA11 divjega tipa s pomočjo mestnospecifične mutageneze uvedli mutacijo D40G, ki je najpogostejši povzročitelj bolezni ALS v evropski populaciji.

Language:Slovenian
Keywords:aneksin A11, amiotrofična lateralna skleroza, izolacija proteina, mutacija D40G
Work type:Bachelor thesis/paper (mb11)
Tipology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2020
COBISS.SI-ID:27959811 Link is opened in a new window
Views:54
Downloads:24
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Secondary language

Language:English
Title:Isolation and characterization of annexin A11 mutants D40G and dN1-118
Abstract:
Amyotrophic lateral sclerosis (ALS) is an untreatable neurodegenerative disease caused by progressive degeneration of motor neurons in the cerebral cortex, brainstem and spinal cord. Up until now, the disease has been associated with mutations in more than 100 genes, including mutations in the ANXA11 gene. Annexin A11 (ANXA11) a Ca2+ regulated phospholipid-binding protein encoded by 505 amino acids that belongs to a larger annexin protein superfamily. ANXA11 is split into two structurally different parts. The C-terminal core contains four annexin domains, which are composed of five alpha helix motifs, in comparison to the extremely variable and disordered N-terminal tail. Because of its intrinsic properties, the structure of the N-terminal tail is yet to be determined. As part of the research work, we prepared shortened versions of the protein; ANXA11dN1-118 had a shortened N-terminal domain by 118 amino acids starting with Ser119 and ANXA11dN-ter starting with Gly179. The sequences for the recombinant proteins were incorporated in bacterial expression vectors pMCSG7 and pMCSG7-GST using ligation independent cloning. We wanted to know whether the GST-tag will increase the protein solubility and reduce its fragmentation, which occurred in previous experiments. Both forms were expressed in the E. coli strain BL21 [DE3] pLysS and isolated using nickel affinity chromatography. We managed to isolate 19,2 mg of ANXA11dN1-118 and 6,4 mg of ANXA11dN-ter protein, which was sufficient for further crystallization experiments. There were no proteolytic cleavages during protein isolation. As part of the research, we also implemented the D40G mutation in ANXA11 wild type protein using site-specific mutagenesis. The mutated form of ANXA11 with D40G mutation is the main cause of ALS in the European population.

Keywords:annexin A11, amyotrophic lateral sclerosis, protein isolation, D40G mutation

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