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

作者：Sharma Sudhir C^*; Armand Tara; Ball K Aurelia; Chen Anna; Pelton Jeffrey G; Wemmer David E; Head Gordon Teresa

来源：Protein Expression and Purification, 2015, 116: 82-89.

DOI：10.1016/j.pep.2015.07.012

• Alzheimer's disease
• Amyloid beta
• 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 beta-amyloid peptides with A beta 42 peptide representing a major isoform in the senile plaques. Given the pathological significance of A beta 42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of A beta 42. This report describes an efficient method to express and purify high quality N-15 isotope-labeled A beta 42 for structural studies by NMR. The protocol involves utilization of an auto induction system with N-15 isotope labeled medium, for high-level expression of A beta 42 as a fusion with IFABP. After the over-expression of the N-15 isotope-labeled IFABP-A beta 42 fusion protein in the inclusion bodies, pure N-15 isotope-labeled A beta 42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled A beta 42 peptide (Garai et al., 2009). We obtain a final yield of similar to 6 mg/L culture for N-15 isotope-labeled A beta 42 peptide. Mass spectrometry and H-1-N-15 HSQC spectra of monomeric A beta 42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with N-15 and C-13 in A beta 42 peptide as well as its other variants including any A beta 42 peptide mutants.

• 出版日期2015-12