Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and fibroblasts. A 15-kDa COOHterminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal growth factor-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. Growth of a human fibroblast cell line, A-1, that required APP for complete growth, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.The characteristic neuropathological features of Alzheimer disease are neuritic plaques containing amyloid and cerebrovascular amyloid deposition (1, 2). Amyloid B-protein, or ,3/A4 protein, a 4-kDa peptide, is a major component of these lesions. fB/A4 protein is derived from a much larger precursor, the amyloid precursor protein (APP). The human APP cDNAs have been cloned, and the APP gene was localized to chromosome 21 (3-6). More than five APP open reading frames have been identified so far (7-11). Three of these-APP-695, APP-751, and APP-770-encode large proteins that have the B/A4 protein sequence near the COOH terminus. Of these three, APP-751 and APP-770 contain a Kunitz-type protease inhibitor domain (7-9). These proteins are posttranslationally glycosylated, tyrosine sulfated, and possibly phosphorylated (12)(13)(14). The proteins are secreted after two proteolytic events-cleavage of the signal peptide (3, 15) and a cleavage within the 8/A4 region (16,17). The pathological process leading to amyloid formation is unknown. Structural and functional analysis of these proteins are hindered by the lack of an abundant source.Prokaryotic expression systems serve as a source of large amounts of proteins; however, these proteins are not posttranslationally modified. Most mammalian expression systems, on the other hand, produce posttranslationally modifled proteins but in insufficient quantities for large-scale production. The baculovirus-infected insect-cell expression system (18) provides a middle ground between these two types of systems. The level of protein production is high, and the proteins produced are post...