Journal: Nature Communications

Article Title: Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase

doi: 10.1038/ncomms3529

Figure Lengend Snippet: γ-Secretase substrates were expressed as fusion proteins with the APP signal peptide and the Profinity eXact tag (Bio-Rad) in sf9 cells ( a ). The Profinity eXact tag protein purification system offers purification of recombinant proteins with a native amino terminus. Once Profinity eXact-tagged γ-secretase substrates were captured using S189 subtilisin-immobilized resin, addition of sodium fluoride triggered precise cleavage at the carboxyl terminus of the cleavage-recognition sequence (shown in green) and the release of substrates with a bona fide N terminus. Purified substrates were recaptured using anti-FLAG M2 agarose beads (Sigma) and eluted by addition of 0.2 M glycine, pH 2.7 and 0.3% NP40. 1/10th volume of 3 M Tris-HCl, pH 8.0 was added to the eluted substrates for neutralization. The flanking region of the S189 subtilisin cleavage site in each substrate is shown in b .

Article Snippet: The cell suspension was mixed with an equal amount of lysis buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 2% NP40 and 2 × protease inhibitor cocktail (Roche Diagnostics)) and incubated on ice for 1 h. After ultracentrifugation at 100,000 × g centrifugation for 1 h, the supernatant was agitated overnight with Profinity eXact (S189 subtilisin-immobilized) resin (Bio-Rad).

Techniques: Protein Purification, Purification, Recombinant, Sequencing, Neutralization