Figure 5

Production of the human γ-secretase components.

(a) Schematic model of the γ-secretase complex. The stars on PS1 indicate the positions of the catalytic residues (D257 and D385 in human PS1). (b) SDS-PAGE images of the T4L-PS1∙Pen-2 complex, produced by the P-MF method and purified by the tag-affinity method with the FLAG-tag at the N-terminus of Pen-2, detected by Coomassie Brilliant Blue staining and western blotting with HisProbe. (c) The enzymatic activity of the T4L-PS1∙Pen-2 complex with the intramolecularly quenched fluorogenic peptide probe. The fluorescence intensities with (triangles) and without (circles) the GXGD protease inhibitor, (Z-Leu-Leu)₂ ketone, at 0–420 min were plotted in this graph. (d) Rapid protein-folding assay, using GFP for the cell-free production of PS1 and the PS1NTF and PS1CTF fragments. The GFP fluorescence was observed with an ImageQuant LAS4000 system (GE Healthcare Life Sciences). (e) Conceptual diagram of the cell-free production of PS1NTF with the addition of purified PS1CTF or Pen-2 to the cell-free reaction solution. (f,g) Cell-free production of T4L-PS1NTF by the P-MF method, with either the coexpression of PS1CTF/Pen-2 or the addition of the purified proteins. (f) Cell-free-produced proteins in the membrane fractions were detected by western blotting with HisProbe-HRP. (g) Densitometry analysis of the bands of the cell-free-produced T4L-PS1NTF in (f) with means ± S.D. (error bars); n = 3. (h) Cell-free production of T4L-PS1NTF by the S-MF method, with either the coexpression of PS1CTF/Pen-2 or the addition of the purified proteins. The T4L-PS1NTF∙PS1CTF complex was purified with the FLAG-tag at the C-terminus of PS1CTF and the T4L-PS1NTF∙Pen-2 complex was purified with the StrepII-tag at the C-terminus of Pen-2. The purified samples were detected by SDS-PAGE and Coomassie Brilliant Blue staining. Gel images are representative of at least two experiments, except for (d). All gel images in this figure were processed by cropping.