Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties.
The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.
Upjohn Laboratories, Kalamazoo, Michigan 49001.
D E
DE
Lowery
J M
JM
Pasternack
P A
PA
Gonzalez-DeWhitt
H
H
Zürcher-Neely
C C
CC
Tomich
R A
RA
Altman
M B
MB
Fairbanks
R L
RL
Heinrikson
S G
SG
Younkin
B D
BD
Greenberg
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011480:Protease Inhibitors; D013379:Substrate Specificity
D016564:Amyloid beta-Protein Precursor; D011480:Protease Inhibitors