A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's β-amyloid peptides from E. coli for NMR-based structural analysis
| Autor: | David E. Wemmer, Jeffrey G. Pelton, K. Aurelia Ball, Anna Chen, Sudhir C. Sharma, Teresa Head-Gordon, Tara Armand |
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| Rok vydání: | 2015 |
| Předmět: |
Gene isoform
chemistry.chemical_classification Amyloid beta-Peptides Nitrogen Isotopes Chemistry Recombinant Fusion Proteins Peptide Mass spectrometry Fatty Acid-Binding Proteins Fusion protein Inclusion bodies Peptide Fragments Article Biochemistry Isotope Labeling Escherichia coli Humans Senile plaques Polyacrylamide gel electrophoresis Nuclear Magnetic Resonance Biomolecular Heteronuclear single quantum coherence spectroscopy Biotechnology |
| Zdroj: | Protein expression and purification. 116 |
| ISSN: | 1096-0279 |
| Popis: | Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants. |
| Databáze: | OpenAIRE |
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