A double cistronic coexpression vector based on the bacterial expression vector pACYCDuet-1 (Novagen) was constructed. The coexpression vector contains the genes for MAβ [MAβ(1–40), MAβ(1–42), or MAβ(1–42)E22G] and (His)₆-tagged Z_Aβ3 in the following order: T7 promoter-1 – MAβ – T7promoter-2 – (His)₆Z_Aβ3 – T7 terminator. MAβ is effectively overexpressed and obtained in the soluble fraction of cell lysates, indicating that its complex with the disulfide-linked Z_Aβ3 dimer is formed and stable in the E. coli cytosol (Figure 2, lane 2).

In the present system, the auxiliary protein Z_Aβ3 is (His)₆-tagged but not the target peptide, permitting the purification of tag-free MAβ without a cleavage reaction. MAβ is captured in complex with Z_Aβ3 by immobilized metal ion affinity chromatography (IMAC) (Figure 2, lane 4), demonstrating that the MAβ:Z_Aβ3 complex remains stable during the initial purification steps. Resonances in the ¹⁵N heteronuclear single quantum correlation (HSQC) NMR spectrum of the coexpressed MAβ(1–40):Z_Aβ3 complex coincide with those of the native Aβ(1–40):Z_Aβ3 complex, indicating that their structures are identical (Figure 3). MAβ is not detected in the IMAC wash fraction (Fig. 2, lane 3). Dissociation of the complex during IMAC is consequently not limiting the peptide yield.

Separation of the MAβ:Z_Aβ3 complex is achieved by IMAC under denaturing conditions. Pure monomeric MAβ is subsequently obtained by application of the denatured peptide to size exclusion chromatography (SEC) using native running buffer, e.g., 20 mM sodium phosphate, 50 mM sodium chloride, pH 7.2.

SDS-PAGE shows a single band corresponding to the monomeric peptide in the case of purified MAβ(1–40), whereas two additional bands at higher molecular weight, approximately at 12 and 15 kDa, are observed for MAβ(1–42) (Figure 2). These bands have been observed before and have been attributed to the SDS-induced formation of Aβ(1–42) oligomers 436. Mass spectrometry confirmed that the bands consist of MAβ(1–42).

The peptide yield from a 1 L culture was 4 mg of MAβ(1–40) or 3 mg of MAβ(1–42). Purification of Z_Aβ3 from an MAβ(1–40) coexpression culture gave 23 mg of the dimeric protein per 1 L of culture, indicating that ~60% of the expressed Z_Aβ3 was in complex with MAβ(1–40), whereas the rest remained unbound.

Several different techniques were used to establish that MAβ and Aβ possess identical conformational properties and aggregation propensities. MAβ and Aβ are indistinguishable by SDS-PAGE (Figure 4A). The extent of SDS-induced oligomer formation of MAβ(1–42) is the same as for Aβ(1–42) and increases with temperature.

In SEC, which separates molecules based on their hydrodynamic volume, very similar elution volumes are obtained for MAβ and Aβ (Figure 4B). The elution volumes correspond to a molecular weight of ~11 kDa on a scale calibrated with globular protein standards, in agreement with previous SEC studies 37. The high apparent molecular weight (the nominal weights of the Aβ and MAβ peptides are in the range of 4.3 to 4.7 kDa) is expected for a peptide that is disordered and consequently has a larger hydrodynamic volume than a globular protein of the same molecular weight.

The secondary structure content was analyzed by circular dichroism (CD) spectroscopy (Figure 4C). Far-UV CD spectra of MAβ conformed to those of Aβ, featuring a minimum at ~198 nm that is characteristic of the predominantly random coil conformation detected in non-aggregated Aβ peptides 3839.

The ¹⁵N HSQC NMR spectra of MAβ(1–40) and MAβ(1–42) strongly resemble those of Aβ(1–40) and Aβ(1–42), respectively (Figure 5). The large majority of Aβ backbone amide resonances are recovered at identical positions in the MAβ spectra. Differences in chemical shifts are only observed for residues N-terminal of Arg5. Such local shift changes are a mandatory consequence of the modification of the peptide sequence, in this case with the N-terminal methionine, and reflect local changes in the electronic environment. However, the chemical shift differences do not demonstrate any change in peptide conformation. The ¹⁵N HSQC spectra prove that Met35 is unoxidized, by comparison with reference spectra for Aβ(1–40) and Aβ(1–42) 35, in agreement with the mass spectrometry results.

The enhanced fluorescence emission of the dye thioflavin T upon binding to amyloid fibrils is frequently used to monitor fibrillation 40. The fibrillation kinetics of Aβ(1–42) and MAβ(1–42) are identical within the error of the experiment (Figure 4D). The presence of amyloid fibrils in the final steady-state of fibrillation was confirmed by electron microscopy (Figure 4E and 4F).

The Arctic mutant of Aβ is a particularly interesting variant inasmuch as it links an increased tendency for protofibril formation and fibrillation to early onset familial AD 89. To our knowledge, no protocol for the recombinant production of Aβ(1–42)E22G has been reported to date, possibly due to the extreme aggregation propensity of this peptide variant. Coexpression of Z_Aβ3 permitted the production of MAβ(1–42)E22G with a yield of 1 mg from a 1 L culture. SDS-PAGE demonstrates increased oligomerization of MAβ(1–42)E22G compared to MAβ(1–42) (Figure 6A). The major fraction of purified MAβ(1–42)E22G is present in monomeric form as evidenced by SEC, which gives an elution volume of ~14 mL (Figure 6B), similar to that of monomeric MAβ(1–40) and MAβ(1–42) (Figure 4B). Recombinant MAβ(1–42)E22G can be employed, e.g., for NMR spectroscopy. The ¹⁵N HSQC NMR spectrum of MAβ(1–42)E22G is displayed in Figure 6C. As expected, the resonances of backbone amides in the vicinity of residue 22 are affected by the E22G mutation due to the removal of one negative charge. However, the changes are not large and the E22G mutant is also disordered in its monomeric state.

The bacterial expression vector pACYCDuet-1 (Novagen) is designed for the double cistronic coexpression of two target genes and contains two multiple cloning sites (MCS), each of which is preceded by a T7 promoter/lac operator and a ribosome binding site. pACYCDuet-1 encoding Aβ(1–40) with an additional N-terminal methionine, cloned as a NcoI/HindIII fragment at MCS 1, was obtained from GENEART. The expression plasmid pAY 442 encoding (His)₆-tagged Z_Aβ3 30 was digested with NdeI and Bpu1102I (all enzymes supplied by New England Biolabs), followed by insertion of the (His)₆-Z_Aβ3 gene into MCS2 of pACYCDuet-1 at the respective restriction sites. The resulting coexpression vector contains the genes in the following order: T7 promoter-1 – MAβ(1–40) – T7promoter-2 – (His)₆Z_Aβ3 – T7 terminator. The vectors for coexpression of MAβ(1–42) and MAβ(1–42)E22G were generated by site-directed mutagenesis (Stratagene QuikChange mutagenesis kit) of the MAβ(1–40) or MAβ(1–42) expression clones, respectively.

BL21(DE3) E. coli cells (Novagen) were transformed with the expression vectors and grown for ~16 h at 37°C on LB agar plates containing 34 μg/mL chloramphenicol. Single colonies were picked and grown for ~16 h in 20 mL ¹⁵N-labeled M9 medium, containing 1 g/L ¹⁵NH₄Cl, 2 g/L glucose, 2 mM MgSO₄, 0.1 mM CaCl₂, 2 g/L natural ¹⁵N-Celtone powder (Spectra Stable Isotopes) and 34 μg/mL chloramphenicol. The pre-culture was transferred to 1 L of ¹⁵N-labeled M9-Celtone medium in a 5 L baffled Erlenmeyer flask. The culture was grown at 37°C with shaking and induced at OD600 ~0.8 by the addition of IPTG to a final concentration of 1 mM. After further growth for 4 hours the cells were harvested and frozen at -20°C. If isotopic labeling was not required, TB medium was used as an alternative to M9-Celtone.

The cell pellet from 1 L of bacterial culture was thawed in an ice/water bath, resuspended in 15 mL of buffer A (50 mM sodium phosphate, 0.2 M sodium chloride, 1 mM PMSF, pH 7.2) and subjected to three freeze-thaw cycles, followed by sonication according to a standard protocol. The lyzed cells were clarified by centrifugation at 17,000 g in a JA 25.50 rotor (Beckman) at 4°C for 30 min.

For capture of the MAβ:Z_Aβ3 complex by IMAC, the supernatant was added to 10 mL HIS-Select Ni²⁺ affinity gel (Sigma-Aldrich) equilibrated in buffer A, and the mixture was incubated batch wise on a roller shaker for 20 min at room temperature. Proteins not bound to the resin were separated by centrifugation at 700 g for 5 min on a swing-out rotor. The resin was washed twice with 50 mL of buffer A, transferred to a 1.5 cm diameter Econo-Column chromatography column (Bio-Rad Laboratories) and washed with another 50 mL of buffer A and 50 mL of buffer A supplemented with 10 mM imidazole. To separate MAβ from the resin-bound Z_Aβ3, the drained resin was resuspended in 40 mL of buffer SL (buffer A supplemented with 6 M guanidine hydrochloride (GdmCl), pH 7.2) and incubated for 30 min at room temperature. The denatured MAβ peptide was recovered in the filtrate upon filtration of the resin slurry through the Econo-Column chromatography column.

Native MAβ was obtained by SEC of the denatured MAβ on a Superdex 75 HR 10/300 column (GE Healthcare) equilibrated with 20 mM sodium phosphate, 50 mM sodium chloride, pH7.2. If protein concentration or storage was desired, the pH of the SEC eluate was adjusted to basic pH (~10.5) directly after elution, as basic pH preserves the monomeric state of Aβ and is thus advantageous for stock solutions 3854. Concentration of the basic protein solutions was achieved using Vivaspin concentration columns (Sartorius). The identity of the peptides was verified by mass spectrometry (MAβ(1–40), theoretical mass: 4458.2 Da, experimental mass: 4458.1 Da; MAβ(1–42), theoretical mass: 4642.3 Da, experimental mass: 4642.3 Da). Peptide concentrations in solution were measured by UV spectroscopy (ε₂₈₀–ε₃₀₀ = 1424 M^-1 cm^-1).

For analysis of the coexpressed MAβ:Z_Aβ3 complex, the entire complex (i.e. without separation of the complex's constituents under denaturing conditions) was eluted from the IMAC column with buffer A supplemented with 150 mM imidazole, and subjected to SDS-PAGE (Figure 2, lane 4). NMR measurements on the complex (Figure 3) were carried out after an additional SEC step using a Superdex 75 HR 10/300 column equilibrated with 20 mM potassium phosphate, pH 7.2.

Aβ was obtained from a commercial source (rpeptide). Aβ(1–40) was purchased either NaOH pre-treated or HFIP pre-treated, dissolved in 30 mM ammonium hydroxide to a concentration of 0.5 mM, and diluted into the final experiment buffer. Aβ(1–42) was purchased HFIP pre-treated. To ensure disaggregation of Aβ(1–42), the peptide was dissolved in 6 M GdmCl (buffer SL) and subjected to SEC, using the same conditions as employed in the final step of the MAβ purification protocol.

Peptides at a concentration of 40–100 μM were analyzed on a Superdex 75 HR 10/300 column equilibrated in 20 mM sodium phosphate, 50 mM sodium chloride, pH 7.2. Elution profiles were normalized to unity at maximum absorbance for the purpose of comparison.

Far-UV CD measurements were performed on a JASCO J-810 spectropolarimeter using a 0.1 cm path length cuvette. Peptides were used at concentrations of 20–25 μM in 20 mM phosphate, pH 7.2–7.4. Spectra were recorded at 20°C. Thirty scans were averaged without smoothing and corrected for the buffer spectrum.

Samples were applied to formvar/carbon coated nickel grids, stained with 2% (w/v) uranyl acetate and viewed in a LEO 912 AB Omega transmission electron microscope.

NMR was measured using Varian Inova 800 MHz and 900 MHz spectrometers. Samples of the MAβ:Z_Aβ3 complex prepared after IMAC and SEC purification contained 160 μM complex in 20 mM potassium phosphate, pH 7.2, with 10% D₂O. Samples of purified MAβ and commercial Aβ peptides contained ca. 60 μM ¹⁵N-labeled peptides in 20 mM sodium phosphate, 50 mM sodium chloride, pH 7.2, with 10% D₂O. NMR data were processed using NMRpipe 55 and analyzed using CcpNmr Analysis 56.

Thioflavin T fluorescence was recorded in 96-well plates (Nunc) using a FLUOstar Optima reader (BMG) equipped with 440 nm excitation and 480 nm emission filters. The samples contained 100 ml of 25 μM MAβ(1–42) or Aβ(1–42) in 20 mM Na-phosphate, 50 mM sodium chloride, pH 7.2, supplemented with 10 μM thioflavin T. Plates were sealed with polyolefin tape (Nunc) and incubated at 37°C. Data points were recorded every 10 min with 50 sec of orbital shaking (width 5 mm) preceding each measurement.