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. 2009 Mar;276(5):1266-81.
doi: 10.1111/j.1742-4658.2008.06862.x.

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

Dominic M Walsh  1 , Eva Thulin, Aedín M Minogue, Niklas Gustavsson, Eric Pang, David B Teplow, Sara Linse
Affiliations

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

Dominic M Walsh et al. FEBS J. 2009 Mar.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Aβ primary sequence and primers used to construct an Aβ synthetic gene. The amino acid sequence of Aβ(M1–40) is shown, with the disease-associated amino acid substitutions indicated above the residues that are replaced. The E. coli-optimized DNA sequence shown below the corresponding amino acids, and the primers used to generate the synthetic gene are indicated by arrows (full sequences are given in Experimental procedures).
Fig. 2
Fig. 2
Aβ(M1–40) and Aβ(M1–42) are expressed in inclusion bodies. (A–D) Pellets of bacteria expressing Aβ(M1–40) (A,B) or Aβ(M1–42) (C,D) were subjected to three rounds of sonication in buffer, and at the end of each sonication step the suspension was centrifuged and the supernatants (labeled S1, S2 and S3) were stored pending analysis. The pellet was then extracted in 8 m urea (fraction labeled U), and purified by ion exchange (fraction labeled IE), filtration through a 30 kDa molecular mass cut-off filter (fraction labeled 30) and concentration on a 3 kDa molecular mass cut-off filter (fraction labeled 3). All fractions were electrophoresed on 10–20% polyacrylamide Tris-tricine gels (A,C) and 1% agarose gels (B,D), and proteins were visualized by Coomassie stain. Lanes HS and LS are molecular mass standards, with the molecular mass in kDa given on the left. (E) 1% agarose gel electrophoresis of urea extracts of inclusion bodies from bacteria expressing Aβ(M1–40) with wild-type (wt) sequence or with the following point mutations: A21G, E22G, E22K, E22Q and D23N. The net charge of each peptide is indicated underneath each lane.
Fig. 3
Fig. 3
Ion-exchange purification of urea-solubilized inclusion bodies. Anion-exchange chromatography in batch mode was performed for Aβ(M1–40) (A,B) and Aβ(M1–42) (C,D). All fractions were electrophoresed on 10–20% polyacrylamide Tris-tricine gels (A,C) or 1% agarose gels (B,D), and proteins were visualized by Coomassie stain. S, combined supernatants after sonication and centrifugation; U, urea-solubilized pellet after third sonication; F, flow-through from application to ion-exchange resin. The peptides were eluted using a stepwise increase in NaCl concentration, and the fractions are labeled as follows: lane 0, 0 mm; lane 1, 50 mm; lane 2, 75 mm; lane 3, 100 mm; lane 4, 125 mm; lane 5, 150 mm; lane 6, 200 mm; lane 7, 250 mm; lane 8, 300 mm; lane 9, 500 mm NaCl. HS and LS, high and low molecular mass standards with the molecular mass in kDa given on the left.
Fig. 4
Fig. 4
LC-MS analysis of bacterially expressed Aβ(M1–40) (B) confirms the correct molecular mass and indicates that the peptide is of comparable purity to synthetic Aβ(1–40) (A). In each panel, the top panel is the HPLC chromatogram obtained with UV absorption at 214 nm, the middle panel is the corresponding total ion-current after infusion into the mass spectrometer, and the bottom panel is the mass spectrum of the major peak observed.
Fig. 5
Fig. 5
Recombinant and synthetic peptides are highly pure and behave similarly on SDS-PAGE and HPLC. Peptides were isolated by SEC and analyzed by reverse-phase HPLC [(A) Aβ(1–40), (B) Aβ(1–42), (C) Aβ(M1–40) and (D) Aβ(M1–42)] and SDS-PAGE (E). Samples electrophoresed on 10–20% polyacrylamide Tris-tricine gels were detected by silver staining. Monomeric Aβ is indicated by an arrow and an Aβ42 species migrating at approximately 14 kDa is indicated by an arrow and an asterisk.
Fig. 6
Fig. 6
Recombinant and synthetic Aβ peptides exhibit similar amyloid-forming properties. Amyloid fibrils and protofibrils bind to ThT, causing a red shift in the excitation spectrum of this compound. A change in the ThT fluorescence at 480 nm was therefore used to monitored the kinetics of amyloid fibril formation by Aβ(1–40) (A), Aβ(M1–40) (B), Aβ(1–42) (E) and Aβ(M1–42) (F). As Aβ fibrillogenesis is known to be highly concentration-dependent, aggregation was monitored both at 6 μm (diamonds, solid line) and 9 μm (triangles, dashed line). Each data point is the mean of eight replicates ± the standard error; where error bars are not visible, the standard error was smaller than the size of the symbols. In all cases, aggregation exhibits a lag phase, subsequent growth and a final equilibrium phase, and the curves shown were fitted to the data by the Boltzmann equation using origin pro 7.5 software (Northampton, MA, USA). The experiment shown is representative of two identical experiments. For electron microscopy, peptide solutions were incubated at 50 μm for 5 h (Aβ40) or 80 min (Aβ42). Triplicate grids for each peptide at each time point were prepared and viewed. The images shown are for Aβ(1–40) (C), Aβ(M1–40) (D), Aβ(1–42) (G) and Aβ(M1–42) (H). Scale bar = 500 nm.
Fig. 7
Fig. 7
Recombinant Aβ peptides inhibit MTT reduction and cause neuronal loss. Monomeric Aβ peptides were isolated by SEC and incubated at 37 °C with shaking until half-maximal aggregation was observed. Peptides were then diluted into neurobasal medium and incubated with neurons at final concentrations of 1, 3 and 6 μm for 6 h. At the end of this period, MTT was added and cells were incubated for a further 2 h. The results are percentage inhibition of MTT reduction relative to control neurons not treated with peptide, and are the mean of three replicates ± standard deviation. (A) Aβ(1–40) (open triangle) and Aβ(M1–40) (inverted open triangle); (B) Aβ(1–42) (closed triangle) and Αβ(M1–42) (inverted closed triangle). To assess the effect of prolonged incubation with Aβ peptides on cell viability, neurons were incubated with 10 μm Aβ(1–40), Aβ(M1–40), Aβ(1–42) or Αβ(M1–42) for 4 days, fixed and then stained with anti-MAP-2 antibody, viewed by light microscopy using a 40× objective lens and photographed (C). The images shown are at a magnification of approximately 200×.
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