Purification of Recombinant α-synuclein : A Comparison of Commonly Used Protocols
The initial state of the intrinsically disordered protein α-synuclein (aSyn), e.g., the presence of oligomers and degradation products, or the presence of contaminants and adducts can greatly influence the aggregation kinetics and toxicity of the protein. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from Escherichia coli: boiling, acid precipitation, ammonium sulfate precipitation, and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using nondenaturing electrospray ionization mass spectrometry, that aSyn isolated by acid precipitation and periplasmic lysis was the purest and yielded the highest percentage of monomeric protein, 100% and 96.5%, respectively. We then show that aSyn purified by the different protocols exerts different metabolic stresses in cells, with the more multimeric/degraded and least pure samples leading to a larger increase in cell vitality. However, the percentage of monomeric protein and the purity of the samples did not correlate with aSyn aggregation propensity. This study highlights the importance of characterizing monomeric aSyn after purification, as the choice of purification method can significantly influence the outcome of a subsequent study.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:59 |
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| Enthalten in: |
Biochemistry - 59(2020), 48 vom: 08. Dez., Seite 4563-4572 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Stephens, Amberley D [VerfasserIn] |
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| Links: |
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| Themen: |
Alpha-Synuclein |
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| Anmerkungen: |
Date Completed 05.04.2021 Date Revised 01.02.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acs.biochem.0c00725 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM318043955 |
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| 500 | |a Date Revised 01.02.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a The initial state of the intrinsically disordered protein α-synuclein (aSyn), e.g., the presence of oligomers and degradation products, or the presence of contaminants and adducts can greatly influence the aggregation kinetics and toxicity of the protein. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from Escherichia coli: boiling, acid precipitation, ammonium sulfate precipitation, and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using nondenaturing electrospray ionization mass spectrometry, that aSyn isolated by acid precipitation and periplasmic lysis was the purest and yielded the highest percentage of monomeric protein, 100% and 96.5%, respectively. We then show that aSyn purified by the different protocols exerts different metabolic stresses in cells, with the more multimeric/degraded and least pure samples leading to a larger increase in cell vitality. However, the percentage of monomeric protein and the purity of the samples did not correlate with aSyn aggregation propensity. This study highlights the importance of characterizing monomeric aSyn after purification, as the choice of purification method can significantly influence the outcome of a subsequent study | ||
| 650 | 4 | |a Comparative Study | |
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| 650 | 4 | |a Journal Article | |
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| 700 | 1 | |a Matak-Vinkovic, Dijana |e verfasserin |4 aut | |
| 700 | 1 | |a Fernandez-Villegas, Ana |e verfasserin |4 aut | |
| 700 | 1 | |a Kaminski Schierle, Gabriele S |e verfasserin |4 aut | |
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