Abstract
α-Synuclein (αS) is an abundant neuronal protein which has been implicated, among others, in the pathogenesis of neurodegenerative diseases like Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). In fact, αS is the major constituent of Lewy bodies, the primarily proteinaceous inclusions found in the nervous tissue of PD and DLB patients. While its physiological role is unclear, it is believed to be involved in the regulation of synaptic vesicle exocytosis. However, in a disease state, αS will “misfold” and aggregate, leading to neuronal dysfunction and death. The study of the molecular events underlying pathogenesis, especially with biophysical and biochemical approaches, requires highly pure untagged αS. In this protocol we describe a method to purify untagged recombinant αS, which can be used for binding, folding, and aggregation studies. The purification method includes a cell lysis step, followed by two chromatography steps: ion-exchange chromatography first, and size-exclusion chromatography for polishing.
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References
Giasson BI, Uryu K, Trojanowski JQ, Lee VMY (1999) Mutant and wild type human α-synucleins assemble into elongated filaments with distinct morphologies in vitro. J Biol Chem 274:7619–7622. https://doi.org/10.1074/jbc.274.12.7619
Mierendorf RC, Novy RE, Morris BB, Hammer B (1998) Expression and purification of recombinant proteins using the pET system. Methods Mol Med 13:257–292
Ren G, Wang X, Hao S et al (2007) Translocation of alpha-synuclein expressed in Escherichia coli. J Bacteriol 189:2777–2786. https://doi.org/10.1128/JB.01406-06
Volles MJ, Lansbury PT (2007) Relationships between the sequence of α-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity. J Mol Biol 366:1510–1522. https://doi.org/10.1016/j.jmb.2006.12.044
GE (2016) Size exclusion chromatography columns and media—selection guide. https://doi.org/10.1111/j.1467-8330.1974.tb00606.x
Pope B, Kent HM (1996) High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res 24:536–537. https://doi.org/10.1093/nar/24.3.536
GE Healthcare Bio-Sciences (2009) ÄKTAprime™ plus Operating Instructions. p 59
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Powers, A.E., Patel, D.S. (2019). Expression and Purification of Untagged α-Synuclein. In: Bartels, T. (eds) Alpha-Synuclein. Methods in Molecular Biology, vol 1948. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9124-2_20
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DOI: https://doi.org/10.1007/978-1-4939-9124-2_20
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9123-5
Online ISBN: 978-1-4939-9124-2
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