A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide

We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (A beta), together with a scaleable and inexpensive purification procedure. A beta(1-40) and A beta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-A beta(1-40) and Met-A beta(1-42), referred to as A beta(M1-40) and A beta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure A beta peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized A beta 1-40 and A beta 1-42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.