Express FP Abeta and lyse cells and wash over beta amyloid?
Membrane Localization of all associated Proteins
Transfect HEK cells with BCR genes (gantenerumab heavy & light chains, CD79A, CD79B), and Lyn. Tage each component with FP. Check for membrane localization
Proper protein Assembly in HEK cells
Transfect HEK cells with BCR genes, Lyn, Syk WT & Protein Kinase domain mutation. Culture both Syk types wit hand without AB and ELISA for phosphorylated Syk and/or Lyn ADD FP Fusions
Recruitment of Syk to the BCR
Transfect HEK cells with BCR genes, Lyn tagged with BFP, Syk tagged with RFP. Culture with and without Beta Amyloid. Check for membrane localization.
TEV Protease Cleavage
Transfect HEK cells with BCR genes, Lyn, and Syk. Fuse TEV cleavage sites with different fluorescent proteins to the heavy chain, CD79A, and CD79B. Fuse TEV protease to Lyn or Syk. Culture with and without Beta Amyloid and check for membrane delocalization.
Full system operation
Transfect HEK cells with BCR genes, Lyn, and Syk. Fuse TEV cleavage sites with TF to all/most promising BCR proteins, Fuse TEV protease to Lynor Syk. Transfect TF promoter expressing RFP. Culture with and without Beta Amyloid and check for flourescence. Characterize signal strength based off of flourescence.
Proteins needed
CD79A
normal
fusion: CD79A C terminus + tev cut site + FP
CD79B
normal
fusion: CD79B C terminus + tev cut site + FP
Lyn
normal
fusion: FP tagged (N or C terminus?)- Do we need this?
point mutation: mutated to always phosphorylate
Syk
fusion: Syk + tev protease, point mutated to prevent phosphorylation , point mutation: FP attached(N or C terminus?), prevent phosphorylation
fusion, point mutatino: tev protease attached (N or C terminus?), point mutated to prevent phosphorylation
ig igM k
with proper localization tag
igM heavy
with proper constant region for membrane localization and proper localization tag