Figure 1
From: Efficient production of a mature and functional gamma secretase protease
Cloning Strategy to incorporate cDNA encoding γ-secretase components into the MultiBac pFBDM plasmid. PS1 and APH1aL cDNAs were cloned from pGEM®T vectors. PEN-2 and Nicastrin DNA constructs were cloned into the pUCS and pSPL vectors respectively. The polH and p10 annotations indicate the promoters of the respective baculoviral genes (A) Step 1: PS1 was cloned at multiple cloning site 1 (MCS1) of the pFBDM plasmid to generate pFBDM-PS1. Step 2: APH1aL was cloned into pFBDM-PS1 at the MCS2 to generate pFBDM-PS1-APH1aL. Step 3: PEN-2 cDNA construct was cloned at the SpeI restriction site on pFBDM-PS1-APH1aL. Step 4: The pSPLpolH-NCT-octa-his was cloned into pFBDM-PS1-Aph1aL-Pen-2 generated in step 3 by Cre-lox recombination to generate pFBDM-γ-secretase (B) A map of the pFBDM-γ-secretase used to generate recombinant baculoviruses (C) A schematic of recombinant γ-secretase enzyme expressed in insect cells showing protein purification tags; PEN-2 tagged with Calmodulin Binding Protein (CBP) at the N-terminus (red arrowhead) and NCT expressed with an “octa-histidine tag” at the C-terminus (red circles).
