Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) and were of the highest purity available. Synthetic peptides Aβ(1–40) and Aβ(1–42) were synthesized in the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT, USA), and purified using reverse-phase HPLC. For both synthetic and recombinant Aβ peptides, the correct mass was confirmed by MALDI-TOF MS and LC-MS.
PCR and cloning procedure
Synthetic genes for Aβ(M1–40) and Aβ(M1–42) were designed using E. coli-favored codons preceded by an ATG initiation codon (). The requirement for a start codon adds a methionine residue at the N-terminus; hence, the peptides expressed here are referred to as Aβ(M1–40) and Aβ(M1–42).
The synthetic gene for Aβ(M1–40) was produced by PCR using Pfusion DNA polymerase (Finnzymes, Espoo, Finland) according to the manufacturer’s guidelines and using the following primers: Aβa, 5′-ATGGACGCTGAATTCCGTCACGACTCTGGTTACGAAGTTCACCACCAGAAGCTGGTG-3′; Aβb, 5′-GTTCACCACCAGAAGCTGGTGTTCTTCGCTGAAGACGTGGGTTCTAACAAGGGTGCT-3′; Aβc, 5′-CACAACGCCACCAACCATCAGACCGATGATAGCACCCTTGTTAGAACCCAC-3′; Aβstart, 5′-GCGTAGGGTCGACATATGGACGCTGAATTCCGTCACG-3′; Aβstop, 5′-CCTGCCGAGCTCCTATTACACAACGCCACCAACCATCAG-3′.
The PCR solution was prepared in the buffer supplied with the enzyme, and contained Aβa, Aβb and Aβc at 40 nm
each, and the start and stop primers Aβstart and Aβstop at 600 nm
each, and 200 μm
each of dATP, dCTP, dGTP and dTTP. The product was separated from primers by agarose gel electrophoresis (2% gel). The full-length gene was cut out from the gel, purified using a GFX PCR and gel band purification kit (GE Healthcare, Chalfont St Giles, UK). The gene was digested with Nde
I and Sac
I restriction enzymes and subjected to a second agarose gel electrophoresis (2% gel), and the cleaved product was purified using the GFX PCR and gel band purification kit. The purified cut gene was ligated into PetSac vector (a modified from of Pet3a with Nde
I and Sac
I cloning sites [43
]) that had been previously cleaved by Nde
I and Sac
I, and used to transformed Ca2+
-competent E. coli
cells (ER2566) by heat shock. The transformed cells were spread on LB agar plates containing ampicillin (50 mg·L−1
), single colonies were picked for 2 mL overnight cultures in LB medium containing ampicillin (50 mg·L−1
), and plasmids were prepared using a GFX plasmid purification kit (GE Healthcare) and sequenced.
The gene for Aβ(Μ1–42) was then produced by PCR using the primers Aβstart and Aβ42stop (5′-CCTGCCGAGCTCCTATTAAGCGATCACAACGCCACCAACCATCAG-3′) and a sequence-verified plasmid carrying the Aβ(Μ1–40) gene. This adds Ile41 and Ala42 to the peptide sequence. The PCR product corresponding to the full-length Aβ(Μ1–42) gene was purified as above and ligated into PetSac. In our PCR design, regions encompassing residues 1–6, 12–18, 24–30 and 34–40 were used as primer annealing sites, and the following codons in these regions were altered to achieve more stable duplexes and/or avoid repeat of similar sequences (K16, AAA→AAG; V24, GTT→GTG; K28, AAA→AAG; G38, GGT→GGC; V40, GTT→GTG). Residues 21–23 are mutated in several Alzheimer’s-like familial disorders [44
]. In our design, residues 19–23 are therefore uniquely encompassed by the middle primer, such that only one additional primer is required for the production of synthetic genes bearing Alzheimer’s disease-associated point mutants.
Sequence-verified plasmids from wild-type and each mutant were transformed into Ca2+-competent E. coli cells (BL21 DE3 PLysS Star) by heat shock and spread on LB agar plates containing ampicillin (50 mg·L−1) and chloramphenicol (30 mg·L−1). Single colonies were used to inoculate 50 mL overnight cultures in LB medium with ampicillin (50 mg·L−1) and chloramphenicol (30 mg·L−1). The next morning, 5 mL of overnight culture was transferred to 500 mL day culture (LB medium with 50 mg·L−1 ampicillin and 30 mg·L−1 chloramphenicol). When the density of cells was sufficient to produce an attenuance at 600 nm (D600 nm) of approximately 0.6, protein expression was induced by addition of isopropyl thio-β-d-galactoside. The cells were harvested between 3 and 4 h after induction, dispensed in Millipore (Carrigtwohill, Cork, Republic of Ireland) H2O (12–25 mL H2O per liter culture), and frozen.
To assay and optimize expression levels, test samples of 1 mL cultures were collected for each transformed bacterial culture at various temperatures (30, 37 and 41 °C) and at various times (1, 2, 3, 4, 5 or 6 h) after induction, and using seven different isopropyl thio-β-d-galactoside concentrations ranging from 0.1 to 2.0 mm for induction. The cell suspension was centrifuged at 5400 g and 4 °C for 15 min, the cell pellet was resuspended in H2O (100 μL) and centrifuged again, after which the supernatant was collected and the pellet dissolved in 8 m urea (100 μL). Both the supernatant and urea-solubilized pellet were then analyzed by agarose gel electrophoresis at pH 8.4 and by SDS-PAGE.
The frozen cell pellet from a 4.5 L culture was thawed, sonicated in a total of 100 mL 10 mm
Tris/HCl pH 8.0, 1 mm
EDTA, for 2 min on ice (1/2 horn, 50% duty cycle), and centrifuged for 10 min at 18 000 g
. The supernatant (S1
in ) was removed, and the pellet was resuspended twice in 100 mL 10 mm
Tris/HCl pH 8.0, 1 mm
EDTA, sonicated and centrifuged as above. The third supernatant was removed, and the pellet was resuspended in 50 mL 8 m
urea, 10 mm
Tris/HCl pH 8.0, 1 mm
EDTA, and sonicated as above, resulting in a clear solution. To minimize carbamylation of Aβ, fresh solutions of ice-cold, deionized ACS grade urea were used, and the duration of exposure to urea was limited to less than 12 h.
Purification of Aβ(M1–40) and Aβ(M1–42)
The procedures described here are for 50 mL of urea-solubilized inclusion bodies originating from 4.5 L of culture, but this process can be scaled proportionally for other amounts. The urea-solubilized inclusion bodies (50 mL) were diluted with 150 mL of 10 mm Tris/HCl pH 8.0 containing 1 mm EDTA (buffer A), added to 50 mL DEAE-cellulose equilibrated in buffer A, and gently agitated for 20 min. The slurry was then applied to a Büchner funnel with filter paper on a vacuum glass bottle [alternatively, a Nalgene (Lima, OH, USA) 0.45 μm filter on a vacuum bottle can be used]. Subsequently, the resin was washed with buffer A (50 mL), followed by stepwise elution using 50 mL aliquots of buffer A with 50, 75, 100, 125, 150, 200, 250, 300 and 500 mm NaCl, respectively. Each aliquot was incubated with the resin for 5 min before collection under vacuum. Eluates were analyzed by SDS-PAGE and agarose gel electrophoresis, and fractions with highly pure Aβ were pooled and fractionated by centrifugation through a 30 kDa molecular mass cut-off filter. The washing and elution processes can also be performed as follows: the resin is washed with 50 mL buffer A, and then with 50 mL buffer A with 25 mm NaCl followed by three or four 50 mL aliquots of buffer A with 125 mm NaCl. Using SDS-PAGE, the peptide is then found in the first two (or first three) 125 mm aliquots, which are combined and used for centrifugal filtration.
Ion-exchange chromatography in column mode
Urea-solubilized inclusion bodies (25 mL originating from 2.2 L of bacterial cell culture) were diluted with 150 mL of buffer A and applied to a 50 mL DEAE-cellulose column equilibrated in buffer A. The column was washed with 50 mL buffer A, followed by elution using a linear gradient from 0–300 mm NaCl with a total gradient volume of 500 mL. Fractions were analyzed by electrophoresis on 10–20% polyacrylamide Tris-tricine gels and 1% agarose gels. In a second set of experiments, the column was equilibrated in buffer A containing 8 m urea, and the sample was eluted with a gradient of 0–300 mm NaCl in buffer A containing 8 m urea.
Mass spectrometry, amino acid analysis and sequencing
Amino acid analysis was performed at the Amino Acid Analysis Center, University of Uppsala, Sweden. Sequence analysis was performed using an Applied Biosystems Procise 492 cLC sequenator (Applied Biosystems, Framingham, MA, USA) employing standard Edman chemistry, and MS analysis was undertaken using an LCQDECA LC/MS system (ThermoFinnigan, San Jose, CA, USA). The MS system consisted of a Surveyor HPLC system with a diphenyl 150 × 1.0 mm column (Grace Vydac, Palo Alto, CA, USA) interfaced to an LCQ-DECA electrospray ionization/ion trap mass spectrometer, and eluted using an acetonitrile/trifluoroacetic acid gradient. MALDI-TOF mass spectrometry was performed using a 4700 proteomics analyzer (Applied Biosystems). Samples were dispensed onto a MALDI sample support, and allowed to air-dry prior to addition of matrix solution (4-hydroxy α-cyano cinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid, 25 mm citric acid). All analyses were performed in positive reflector mode, collecting data from approximately 3000 and 5000 single laser shots for MS and MS/MS analyses, respectively.
Preparation of aggregate-free monomer for fibrillation assays
For fibrillation assays, it is essential to start with a uniform monomeric peptide sample. Solutions of monomeric Aβ were prepared by dissolving lyophilized peptides in 5 m GuHCl, Tris/HCl pH 8.0 at a concentration of approximately 1 mg·mL−1, and isolating monomers using SEC. Aβ solutions were chromatographed on a Superdex 75 10/300 GL column using an ÄKTA purifier (GE Healthcare), and eluted at 0.8 mL·min−1 using 50 mm ammonium acetate, pH 8.5. Fractions (0.5 mL) were collected, peak fractions pooled, and the concentration of peptide determined by absorbance at 275 nm using ε275 = 1400 m−1 cm−1.
Assessment of aggregation using thioflavin T binding and electron microscopy
The kinetics of fibril formation was determined using a continuous ThT assay [49
]. Solutions of Aβ isolated by SEC were diluted to concentrations of 36 or 24 μm
using 50 mm
ammonium acetate, pH 8.5. Peptides were then incubated in a 96-well black fluorescence plate at a final concentration of 6 or 9 μm
in the presence of 10 μm
ThT at 37 °C, and shaken at 700 r.p.m. using a VorTemp 56™ incubator/shaker with an orbit of 3 mm (Labnet International, Windsor, UK). Measurements were made at regular intervals using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation and emission at 440 and 480 nm, respectively. Each experimental point is the mean of the fluorescence signal of at least eight wells containing aliquots of the same solution. The morphology of Aβ aggregates formed from solutions incubated as above but in the absence of ThT and at a concentration of 50 μm
was assessed by negative-contrast electron microscopy as described previously [25
]. Briefly, samples were applied to a carbon-coated formvar grid, left for 1 min, fixed with glutaraldehye, wicked dry with filter paper, and 2% uranyl acetate was added and the mixture was incubated for 2 min. The grid was wicked dry and allowed to air dry for 10 min. Samples were stored in a sealed container and viewed under a Tecani G2 BIOTWIN electron transmission microscope operated at 120 V. All reagents were supplied by Electron Microscopy Sciences (Hatfield, PA, USA).
Assessement of SEC-isolated peptides by HPLC and SDS-PAGE
Samples (100 μL) of peptides isolated by SEC were injected on to a CN capcell column (4.6 mm × 25 cm) (Shiseido Fine Chemicals, Toyko, Japan) using a Varian Pro Star 410 autosampler (Varian Inc., Palo Alto, CA, USA), and eluted at 1.5 mL·min−1
with a 14–49% acetonitrile gradient using a Varian Pro Star HPLC system fitted with a photodiode array detector. For SDS-PAGE, samples (10 μL) were mixed with 2× sample buffer, and immediately electrophoresed on 10–20% polyacrylamide Tris-tricine gels. Proteins were stained with silver as described previously [28
Primary hippocampal neuronal cultures were prepared as described previously [30
] with minor modifications. Briefly, primary hippocampal cultures were generated from embryonic day 18 Wistar rats. Hippocampi were dissected out in Hanks’ balanced salt solution buffered with HEPES, and dissociated using papain. Cells were plated at 6 × 104
cells on 48-well dishes pre-coated with poly-d
-lysine (50 μg·mL−1
) and maintained in neurobasal medium containing 2 mm
glutamine and B27 supplement without antioxidants. Half the medium was exchanged every 3 days. All media reagents were purchased from Invitrogen (Dun Laoghaire, Republic of Ireland).
Preparation of peptide for cell treatment
Lyophilized peptides were resuspended and incubated for a minimum of 2 h in 5 m
GuHCl, pH 8.0. Thereafter, samples were injected onto a Superdex 75 column HR 10/30 column (Amersham Biosciences, Amersham, UK), and eluted with 10.9 mm
HEPES pH 7.4 at a flow rate of 0.8 mL·min−1
. Peak fractions were then examined for absorbance at 275 nm, and the concentration of Aβ was calculated. Fractions containing monomeric peptide were diluted such that all peptides were of equal concentration. To induce peptide aggregation, samples were incubated at 37 °C and shaken at 700 r.p.m. using a VorTemp 56™ incubator/shaker with an orbit of 3 mm (Labnet International) until 50% of the maximal thioflavin T fluorescence had been achieved; maximal aggregation was taken as the mean plateau fluorescent signal. Peptides were then diluted with 2× neurobasal medium, and 50% of the medium of each well was replaced with an equal volume of neurobasal medium containing either Aβ(1–40), Aβ(M1–40), Aβ(1–42) or Aβ(M1–42) (1, 3 or 6 μm
, final concentration) and incubated for 6 h. Cell-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was assessed as described previously [30
]. Briefly, following incubation with peptides, 2.5 mg·mL−1
MTT (25 μL) was added to each well, and incubation was continued for a further 2 h. Cells were then solubilized in 250 μL of 20% w/v SDS in 50% v/v N,N′
-dimethylformamide, 25 mm
HCl, 2% v/v glacial acetic acid, pH 4.7, and levels of reduced MTT were determined by measuring the difference in absorbance at 570 and 650 nm using a Molecular Devices Spectramax M2 microplate reader.
In a separate series of experiments, neurons were incubated for 4 days with each of the peptides (10 μm), and cells were fixed and used for immunocytochemical analyses.
Neurons were fixed in 4% paraformaldehyde for 20 min at room temperature, and cells were stained for microtubule-associated protein-2 (MAP-2) using a Vectastain kit (Vector Laboratories, Peterborough, UK). Staining was performed according to the manufacturer’s instructions. Briefly, endogenous peroxidases were blocked in 0.3% H2O2, rinsed in NaCl/Pi and incubated in blocking solution for 20 min (Vectastain). Neurons were then incubated with mouse monoclonal anti-MAP-2 (Sigma, Poole, UK) diluted 1 : 2000 in blocking solution for 30 min. Cells were rinsed in NaCl/Pi several times and incubated in blocking serum containing anti-mouse IgG (Vectastain) for a further 30 min. Staining was developed by incubation of cells with Vectastain ABC reagent for 30 min, followed by incubation with substrate solution until colour had developed. Cells were visualized by light-phase contrast microscopy using a 40× objective lens, and captured using an SP-500 UZ digital compact camera (Olympus, Watford, UK).
Co-expression with Met aminopeptidase
Plasmids encoding MetAP-TG (a mutated form of Met aminopeptidase that can cleave N-terminal Met when the second residues is charged [24
]) and Aβ were electroporated into E. coli
cells (BL21 DE3 PLysS Star) and spread on LB plates with ampicillin, kanamycin and chloramphenicol. Single colonies were picked for cultivation in liquid culture as described for Aβ alone, except that the medium contained 50 mg·L−1
ampicillin, 100 mg·L−1
kanamycin and 30 mg·L−1