α-synuclein is a 140-amino-acid protein that is involved in the onset of Parkinson's disease. Several mutations, mainly located in the N-terminal region, can lead to an increase in its oligomerisation and fibrillation. Aggregation and accumulation of synuclein are associated with neurotoxicity, due in particular to the formation of intracellular amyloidotic deposits of the protein itself, which lead to a high production of reactive oxygen species (ROS) and thus oxidative stress. The following synuclein mutations were analysed: E46K and A30P. Expression of the two mutants of the protein occurs in E. coli BL21 using the pT7-7 cloning vector, which is inserted into the bacteria by chemical transformation. The transformed bacteria are grown in LB medium containing ampicillin and X-gal. For the extraction of the synuclein produced, the bacteria are lysed with the use of Osmotic Shock Buffer solution. Subsequently, the protein is purified using chromatographic techniques such as SDS-PAGE, HPLC and ion exchange FPLC, and characterised from a biophysical point of view using techniques such as mass spectrometry, dynamic light scattering, circular dichroism and UV-Visible absorption.
L’α-sinucleina è una proteina di 140 amminoacidi che è coinvolta nell’insorgenza della malattia di Parkinson. Diverse mutazioni, localizzate soprattutto nella regione N-terminale, possono portare ad un aumento della sua oligomerizzazione e fibrillazione. L’aggregazione e l’accumulo di sinucleina sono associati alla neurotossicità, dovuta in particolare alla formazione di depositi amiloidotici intracellulari della proteina stessa, i quali portano ad una elevata produzione di specie reattive dell’ossigeno (ROS) e quindi stress ossidativo. Sono state analizzate le seguenti mutazioni della sinucleina: E46K e A30P. L’espressione dei due mutanti della proteina avviene in E. coli BL21 mediante l’uso del vettore di clonaggio pT7-7, inserito all’interno dei batteri mediante trasformazione chimica. I batteri trasformati vengono fatti crescere in terreno LB contenente ampicillina e X-gal. Per l’estrazione della sinucleina prodotta i batteri vengono lisati con l’uso della soluzione Osmotic Shock Buffer. Successivamente la proteina viene purificata con tecniche cromatografiche, quali SDS-PAGE, HPLC e FPLC a scambio ionico, e caratterizzata da un punto di vista biofisico mediante tecniche quali spettrometria di massa, dynamic light scattering, dicroismo circolare e assorbimento UV-Visibile.
Caratterizzazione biofisica di proteine
FORNASIERO, MATTEO
2023/2024
Abstract
α-synuclein is a 140-amino-acid protein that is involved in the onset of Parkinson's disease. Several mutations, mainly located in the N-terminal region, can lead to an increase in its oligomerisation and fibrillation. Aggregation and accumulation of synuclein are associated with neurotoxicity, due in particular to the formation of intracellular amyloidotic deposits of the protein itself, which lead to a high production of reactive oxygen species (ROS) and thus oxidative stress. The following synuclein mutations were analysed: E46K and A30P. Expression of the two mutants of the protein occurs in E. coli BL21 using the pT7-7 cloning vector, which is inserted into the bacteria by chemical transformation. The transformed bacteria are grown in LB medium containing ampicillin and X-gal. For the extraction of the synuclein produced, the bacteria are lysed with the use of Osmotic Shock Buffer solution. Subsequently, the protein is purified using chromatographic techniques such as SDS-PAGE, HPLC and ion exchange FPLC, and characterised from a biophysical point of view using techniques such as mass spectrometry, dynamic light scattering, circular dichroism and UV-Visible absorption.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/70525