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 (Abeta), together with a scaleable and inexpensive purification procedure. Abeta(1-40) and Abeta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Abeta(1-40) and Met-Abeta(1-42), referred to as Abeta(M1-40) and Abeta(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 Abeta peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Abeta1-40 and Abeta1-42. 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.
Walsh
Dominic M
DM
Laboratory for Neurodegenerative Research, School of Biomolecular and Biomedical Science, Conway Institute, Belfield, University College Dublin, Republic of Ireland. dominic.walsh@ucd.ie
Thulin
Eva
E
Minogue
Aedín M
AM
Gustavsson
Niklas
N
Pang
Eric
E
Teplow
David B
DB
Linse
Sara
S
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D007150:Immunohistochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)