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)¹ 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 β-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 β-protein domain at Gln¹⁵ or Lys¹⁶, essentially identical to previous reports utilizing mammalian cell systems (1). Rare secreted forms (< 5%) appeared to extend to Lys²⁸. Secretion constructs produced these same forms, but in different ratios. Most (~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 inhibitory 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.