ISOLATION OF BACULOVIRUS-DERIVED SECRETED AND FULL-LENGTH BETA-AMYLOID PRECURSOR PROTEIN

We have expressed two forms of the Alzheimer's beta-amyloid precursor protein (beta-APP), the 695-amino acid form (695-beta-APP), and the 751-amino acid form (751-beta-APP) in a baculovirus system. Both forms were expressed as full-length precursor, and were subsequently processed in vivo to release extracellular secreted proteins. The secreted forms were cleaved from the full-length beta-APP in a manner analogous to the cleavage of beta-APP during constitutive secretions in mammalian cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., Beyreuther, K. (1989) Cell 57, 115-126; Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I., and Fritz, L. J. (1990) J. Biol. Chem. 265, 4492-4497). High levels of expression of 20-50 mg/liter were achieved. Both full-length and secreted forms of the beta-amyloid precursor proteins were purified using a combination of ion-exchange and immunoaffinity chromatography using a monoclonal antibody directed against beta-APP. The 751-beta-APP-derived full-length and secreted forms, which contain the Kunitz protease inhibitor domain, were shown to be as active in the inhibition of trypsin as is mammalian-derived secreted beta-APP. The availability of purified full-length beta-APP from the baculovirus system will be valuable for biochemical and cell biological analyses tha may elucidate the mechanism of the inappropriate processing that leads to beta-amyloid formation in Alzheimer's disease.