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Purification of Recombinant α-synuclein: A Comparison of Commonly Used Protocols.

Accepted version
Peer-reviewed

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Type

Article

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Authors

Stephens, Amberley D  ORCID logo  https://orcid.org/0000-0002-7303-6392
Matak-Vinkovic, Dijana 
Fernandez-Villegas, Ana 
Kaminski Schierle, Gabriele S  ORCID logo  https://orcid.org/0000-0002-1843-2202

Abstract

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.

Description

Keywords

Cell Line, Cell Survival, Chemical Precipitation, Chromatography, Gel, Chromatography, Ion Exchange, Chromatography, Liquid, Escherichia coli, Humans, Intrinsically Disordered Proteins, Microscopy, Electron, Transmission, Protein Aggregates, Protein Conformation, Protein Conformation, beta-Strand, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, alpha-Synuclein

Journal Title

Biochemistry

Conference Name

Journal ISSN

0006-2960
1520-4995

Volume Title

59

Publisher

American Chemical Society (ACS)

Rights

All rights reserved
Sponsorship
Alzheimer's Research UK (ARUK-PG2013-14)
Wellcome Trust (089703/Z/09/Z)
Medical Research Council (MR/N012453/1)
Wellcome Trust (203249/Z/16/Z)
Michael J. Fox Foundation (MJFF) (16238)
Medical Research Council (MR/K02292X/1)
Wellcome Trust (065807/Z/01/Z)
Biotechnology and Biological Sciences Research Council (BB/H023917/1)
Medical Research Council (G0902243)
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