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bioRxiv · 10.1101/2020.05.13.093286

Purification of recombinant α-Synuclein: a comparison of commonly used protocols

Stephens, A. D.·Matak-Vinkovic, D.·Kaminski Schierle, G. S.
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Abstract

The insoluble aggregated form of the protein alpha-synuclein (aSyn) is associated with synucleinopathies, such as Parkinsons Disease, therefore great effort is put into understanding why and how this initially soluble protein misfolds. The initial state of aSyn, e.g. presence of contaminants, adducts, oligomers or degradation products, can greatly influence the outcome of an assay, such as determining its aggregation kinetics. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from E. coli by boiling, acid precipitation, ammonium sulphate precipitation and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using non-denaturing electrospray ionisation mass spectrometry of the differently extracted aSyn samples, that aSyn isolated by acid precipitation and periplasmic lysis yielded the highest percentage of monomer, 100% and 96.5% respectively. aSyn purity was again highest in samples isolated by acid precipitation and periplasmic lysis, yet aggregation assays displayed differences in the aggregation rate of aSyn isolated by all four methods. HighlightsO_LIA rapid protocol; expression day one, two step purification day two. C_LIO_LIThe periplasmic lysis-based protocol yielded 95% pure aSyn. C_LIO_LIAcid precipitation and periplasmic lysis-based protocols yielded the highest proportion of monomeric aSyn at 100% and 96.5%, respectively. C_LI

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Stephens, A. D., Matak-Vinkovic, D., Kaminski Schierle, G. S.. 2020-05-15. Purification of recombinant α-Synuclein: a comparison of commonly used protocols. https://doi.org/10.1101/2020.05.13.093286

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