Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.
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 COOH-terminal 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.
Bhasin
R
R
Department of Psychiatry and Behavioral Science, State University of New York, Stony Brook 11794-8101.
Van Nostrand
W E
WE
Saitoh
T
T
Donets
M A
MA
Barnes
E A
EA
Quitschke
W W
WW
Goldgaber
D
D
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D016367:Baculoviridae / Q000235:genetics*; D002352:Carrier Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007313:Insecta; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D058255:Protease Nexins; D011485:Protein Binding; D012326:RNA Splicing; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins