dc.contributor.author | Kloepper, KD | en_US |
dc.contributor.author | Woods, WS | en_US |
dc.contributor.author | Winter, KA | en_US |
dc.contributor.author | George, JM | en_US |
dc.contributor.author | Rienstra, CM | en_US |
dc.date.accessioned | 2013-01-11T11:44:35Z | |
dc.date.available | 2006-02-14 | en_US |
dc.date.issued | 2006-07 | en_US |
dc.identifier.issn | 1046-5928 | en_US |
dc.identifier.uri | http://qmro.qmul.ac.uk/xmlui/handle/123456789/3134 | |
dc.description.abstract | We report the expression and purification of alpha-synuclein, a protein implicated in Parkinson's disease, from isotopically (13C, 15N) labeled bacterial growth media, as required for solid-state NMR structural studies. Expression from Escherichia coli (BL21(DE3)) was performed with a protocol optimized for time efficiency and yield. Chemical lysis, crude purification by ammonium sulfate precipitation, and two chromatography steps (hydrophobic interaction and size exclusion) yield 30-35 mg/L of growth medium. Purity is confirmed by gel electrophoresis and mass spectrometry. Furthermore, we demonstrate reproducible fibril growth by control of environmental incubation conditions. Highly resolved multidimensional solid-state NMR spectra indicate microscopic order throughout the majority of the AS fibril structure. The number of signals and intensities of well-resolved residue types (Thr, Ser, Ala, Gly, Val, and Ile) are consistent with a single conformation, which is reproducibly prepared by seeding consecutive preparations. Variations in the fibril growth rates and structural polymorphisms exhibited in the solid-state NMR spectra are minimized by careful control of incubation conditions. | en_US |
dc.format.extent | 112 - 117 | en_US |
dc.language | eng | en_US |
dc.language.iso | en | en_US |
dc.relation.ispartof | Protein Expr Purif | en_US |
dc.rights | https://doi.org/10.1016/j.pep.2006.02.009 | |
dc.subject | Carbon Isotopes | en_US |
dc.subject | Escherichia coli | en_US |
dc.subject | Humans | en_US |
dc.subject | Mutation | en_US |
dc.subject | Nitrogen Isotopes | en_US |
dc.subject | Nuclear Magnetic Resonance, Biomolecular | en_US |
dc.subject | alpha-Synuclein | en_US |
dc.title | Preparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms. | en_US |
dc.type | Article | |
dc.rights.holder | Copyright © 2006 Elsevier Inc. | |
dc.identifier.doi | 10.1016/j.pep.2006.02.009 | en_US |
pubs.author-url | https://www.ncbi.nlm.nih.gov/pubmed/16564705 | en_US |
pubs.issue | 1 | en_US |
pubs.notes | Not known | en_US |
pubs.publication-status | Published | en_US |
pubs.volume | 48 | en_US |
dcterms.dateAccepted | 2006-02-14 | en_US |