Purification of Amyloid-Beta Inclusion Bodies

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.