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dc.contributor.authorKloepper, KDen_US
dc.contributor.authorWoods, WSen_US
dc.contributor.authorWinter, KAen_US
dc.contributor.authorGeorge, JMen_US
dc.contributor.authorRienstra, CMen_US
dc.date.accessioned2013-01-11T11:44:35Z
dc.date.available2006-02-14en_US
dc.date.issued2006-07en_US
dc.identifier.issn1046-5928en_US
dc.identifier.urihttp://qmro.qmul.ac.uk/xmlui/handle/123456789/3134
dc.description.abstractWe 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.extent112 - 117en_US
dc.languageengen_US
dc.language.isoenen_US
dc.relation.ispartofProtein Expr Purifen_US
dc.rightshttps://doi.org/10.1016/j.pep.2006.02.009
dc.subjectCarbon Isotopesen_US
dc.subjectEscherichia colien_US
dc.subjectHumansen_US
dc.subjectMutationen_US
dc.subjectNitrogen Isotopesen_US
dc.subjectNuclear Magnetic Resonance, Biomolecularen_US
dc.subjectalpha-Synucleinen_US
dc.titlePreparation of alpha-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms.en_US
dc.typeArticle
dc.rights.holderCopyright © 2006 Elsevier Inc.
dc.identifier.doi10.1016/j.pep.2006.02.009en_US
pubs.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/16564705en_US
pubs.issue1en_US
pubs.notesNot knownen_US
pubs.publication-statusPublisheden_US
pubs.volume48en_US
dcterms.dateAccepted2006-02-14en_US


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