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 secretion in mammalian cells (Weidemann, A., König, 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 that may elucidate the mechanism of the inappropriate processing that leads to beta-amyloid formation in Alzheimer's disease.
Knops
J
J
Athena Neurosciences, Inc., South San Francisco, California 94080.
Johnson-Wood
K
K
Schenk
D B
DB
Sinha
S
S
Lieberburg
I
I
McConlogue
L
L
D016229:Amyloid beta-Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D016564:Amyloid beta-Protein Precursor; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002846:Chromatography, Affinity; D005091:Exons; D006801:Humans; D007700:Kinetics; D011480:Protease Inhibitors / Q000302:isolation & purification; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D011480:Protease Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; D014357:Trypsin