Fig 1.
Expression and purification of fusion Aβ(E3Q-42) protein in E. coli BL21 (DE3) pLysS.
(a) 15% Tris/Glycine-SDS-PAGE analysis of the lysates before IPTG induction (t = 0 h) and after IPTG induction leading to expression of fusion Aβ(E3Q-42) (t = 16 h after induction) at 30°C. (b) Analytical RP-HPLC of IMAC purified fusion Aβ(E3Q-40) (red) and fusion Aβ(E3Q-42) (black) with typical retention times of 5 and 7 min, respectively. (c) 15% Tris/Glycine-SDS-PAGE of IMAC and RP-HPLC purified fusion Aβ(E3Q-42).
Table 1.
Yields of intermediates and final purified pEAβ peptides in mg per l culture broth.
Fig 2.
Analytical RP-HPLC of TEV protease cleavage reaction of Aβ(E3Q-40) (a) and Aβ(E3Q-42) (b).
The peptides were applied on an analytical Zorbax SB-300 C8 column and eluted with 30% ACN and 0.1% TFA at 80°C. Black lines indicate analysis after 0.5 h and red lines after 7 h TEV cleavage reaction at 4°C. Peaks for the fusion proteins decreased while peaks indicating cleaved Aβ(E3Q-40) or Aβ(E3Q-42) were increasing. (c) Semi-preparative RP-HPLC of TEV-cleaved fusion Aβ(E3Q-40) to separate Aβ(E3Q-40) from remaining fusion protein.
Fig 3.
Non-enzymatic pyroglutamate (pE) formation by acidic and elevated temperature conditions of Aβ(E3Q-40) (a), Aβ(E3Q-42) (b) and wild type Aβ(3–40) (c).
Peptides were incubated at 45°C in sodium acetate buffer pH 3.5 for 24 h. Conversion was observed by analytical RP-HPLC with an analytical Zorbax SB-300 C8 column in 30% ACN/ 0.1% TFA at 80°C. Peaks for non-modified peptides decreased while peaks for the pEAβ variants appeared at a longer retention time. (d) Reaction scheme of the conversion of N-terminal E3 or N-terminal Q3 to pE. (e) Mass spectrometry of [U-15N]Aβ(E3Q-40) compared to N-terminally modified [U-15N]pEAβ40. Molecular mass of the peptides differs in 18 Da due to the loss of the 15NH3 group.
Fig 4.
Analytics of final purified pEAβ.
Analytical RP-HPLC of pEAβ40 (a) and pEAβ42 (b) after final purification and corresponding analysis of the proteins by Tris/Tricine-SDS-PAGE (c). The characteristic RP-HPLC retention times are approximately 9.5 min for pEAβ40 and 15 min for pEAβ42.
Fig 5.
ThT assay of 10 μM recombinant pEAβ40 and pEAβ42.
Experiments were performed in 10 mM sodium phosphate buffer pH 7.4 at 37°C. Binding of ThT (10 μM final concentration) to Aβ fibrils was determined by fluorescence at an extinction of 440 nm and emission at 492 nm.
Fig 6.
1H,15N-HSQC spectra of Aβ(E3Q-40) (blue) and pEAβ40 (red) (a) or of pEAβ42 (b).
NMR spectra were recorded from 25 μM protein samples solved in 10 mM sodium phosphate buffer pH 7.4 at 5°C on a 600 MHz Bruker spectrometer. Note that in (a) “blue” signals derived from Aβ(E3Q-40) are overlaid with “red” signals from pEAβ40. Therefore “blue” signals are not visible, if identical in shift and intensity to “red” signals.