A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.
 

A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.

Related Articles A Facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.

Protein Expr Purif. 2015 Jul 28;

Authors: Sharma SC, Armand T, Aurelia Ball K, Chen A, Pelton JG, Wemmer DE, Head-Gordon T

Abstract
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 ?-amyloid peptides with A?42 peptide representing a major isoform in the senile plaques. Given the pathological significance of A?42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of A?42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled A?42 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 A?42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-A?42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled A?42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled A?42 peptide (Protein Expr. Purif. 66 (2009) 107-112). We obtain a final yield of ~6 mg/L culture for (15)N isotope-labeled A?42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric A?42 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 A?42 peptide as well as its other variants including any A?42 peptide mutants.


PMID: 26231074 [PubMed - as supplied by publisher]



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