Abstract
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.
Highlights
COOH-terminal fragments identified in fractions 34 and 52 since they were present at a yield of 50 pmol, as compared with 1.8 nmol for the shorter@-proteinpeptides 1-15 and 1
This work demonstrates the utility of the baculovirus/ insect cell expression system as a source of milligram quantities of recombinant AAP protein for biochemical studies
The baculovirus/insect cell system permits each recombinant human AAP to be produced in large amounts and recovered in pure form without contamination by endogenous forms
Summary
Compositional analysis of proteins and peptides were obtained by conventional automated ion-exchange chromatography on a Beckman model 6300 analyzer. Samples were dried in aSpeed Vac Concentrator (Savant), and threesidueswere dissolved in buffer at pH2.2 (NaS; Beckman) for application to the analyzer. Sequence analyses of AAP proteins and CNBr fragments were performed by automated Edman degradation in an Applied Biosystems Inc. model470 sequenatorfitted with an on-line HPLC analyzer (model 120-A)for phenylthiohydantoin amino acids. Peaks from the latter were integrated by a Nelson Analytical 3000 Series chromatography data system connected in parallel with the recorder to the output of the HPLC system
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