Methodology article

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

Bertil Macao^1* , Wolfgang Hoyer^1* , Anders Sandberg¹ , Ann-Christin Brorsson² , Christopher M Dobson² and Torleif Härd^1,3

¹  Department of Medical Biochemistry, University of Gothenburg, PO Box 440, SE-405 30 Göeborg, Sweden

²  Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

³  The Swedish NMR Centre, University of Gothenburg, Box 465, SE-405 30 Göeborg, Sweden

author email corresponding author email* Contributed equally

BMC Biotechnology 2008, 8:82doi:10.1186/1472-6750-8-82

Published:	30 October 2008

Abstract

Background

Oligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein Z_Aβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. Z_Aβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation.

Results

Coexpression of Z_Aβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40) and Aβ(1–42), yielding 4 or 3 mg, respectively, of pure ¹⁵N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. Z_Aβ3 coexpression moreover permits the recombinant production of Aβ(1–42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Aβ(1–42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies.

Conclusion

The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Aβ(1–42) is reported.