A Facile Method for Expression and Purification of 15N Isotope-labeled Human Alzheimer's β-amyloid Peptides from E. coli for NMR-based Structural Analysis
Alzheimer's disease, amyloid β, fatty acid binding protein, expression, HSQC
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 15N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with 15N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the 15N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure 15N 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 15N isotope-labeled Aβ42 peptide. Mass spectrometry and 1H–15N 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 15N and 13C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.
Protein Expression and Purification
Sudhir C. Sharma, Tara Armand, K. Aurelia Ball, Anna Chen, Jeffrey G. Pelton, David E. Wemmer, Teresa Head-Gordon. A facile method for expression and purification of 15N isotope labeled human Alzheimer’s β-amyloid peptides from E. coli for NMR-based structural analysis. Protein Expression and Purification, 2015; 116:82-89.