Proc. Natl. Acad. Sci. USAVol. 88, pp. 10307-10311, November 1991Biochemistry

Expression of active secreted forms of human amyloid ,8-proteinprecursor by recombinant baculovirus-infected insect cellsRAMANINDER BHASIN*, WILLIAM E. VAN NOSTRANDt, TSUNAO SAITOHO, MIKHAIL A. DONETS*,ELIZABETH A. BARNES*, WOLFGANG W. QUITSCHKE*, AND DMITRY GOLDGABER**Department of Psychiatry and Behavioral Science, State University of New York, Stony Brook, NY 11794-8101; tDepartment of Microbiology and MolecularGenetics, University of California, Irvine, CA 92717; and tDepartment of Neurosciences, University of California, San Diego, School of Medicine, La Jolla,CA 92093-0624

Contributed by D. Carleton Gajdusek, August 6, 1991

ABSTRACT Three alternatively spliced forms of the amy-loid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vectorsystem. The recombinant proteins were secreted into theculture medium by infected insect cells, and APP moleculeswere detected in insect cells and medium 2 days after infectionwith the recombinant APP-baculoviruses. A partial sequence ofthe NH2 terminus of the secreted protein revealed identity withthe native secreted protein and showed that the signal peptidewas recognized and properly cleaved in insect cells. Purifiedsecreted recombinant APP-751 comigrated with protease nexin2 purified from platelets and fibroblasts. A 15-kDa COOH-terminal fragment of APP was also detected in cells infectedwith recombinant baculoviruses, suggesting that recombinantAPP proteins were cleaved at the COOH-terminal end likenative APP protein. Recombinant APP-751 and APP-770formed complexes with epidermal growth factor-binding pro-tein, whereas APP-695 did not. In addition, recombinantAPP-751 and APP-770 inhibited trypsin and chymotrypsinactivity, whereas APP-695 did not. Growth of a human fibro-blast cell line, A-1, that required APP for complete growth, wasrestored upon addition of secreted recombinant APP-695 orAPP-751. Thus, the appropriately sized, secreted recombinantAPP proteins produced in this expression system are biologi-cally active.

The characteristic neuropathological features of Alzheimerdisease are neuritic plaques containing amyloid and cere-brovascular amyloid deposition (1, 2). Amyloid B-protein, or,3/A4 protein, a 4-kDa peptide, is a major component of theselesions. fB/A4 protein is derived from a much larger precur-sor, the amyloid precursor protein (APP). The human APPcDNAs have been cloned, and the APP gene was localized tochromosome 21 (3-6). More than five APP open readingframes have been identified so far (7-11). Three of these-APP-695, APP-751, and APP-770-encode large proteins thathave the B/A4 protein sequence near the COOH terminus. Ofthese three, APP-751 and APP-770 contain a Kunitz-typeprotease inhibitor domain (7-9). These proteins are post-translationally glycosylated, tyrosine sulfated, and possiblyphosphorylated (12-14). The proteins are secreted after twoproteolytic events-cleavage of the signal peptide (3, 15) anda cleavage within the 8/A4 region (16, 17). The pathologicalprocess leading to amyloid formation is unknown. Structuraland functional analysis of these proteins are hindered by thelack of an abundant source.

Prokaryotic expression systems serve as a source of largeamounts of proteins; however, these proteins are not post-translationally modified. Most mammalian expression sys-tems, on the other hand, produce posttranslationally modi-

fled proteins but in insufficient quantities for large-scaleproduction. The baculovirus-infected insect-cell expressionsystem (18) provides a middle ground between these twotypes of systems. The level of protein production is high, andthe proteins produced are posttranslationally modified, al-though they may not be as extensively glycosylated as theirmammalian counterparts because insect cells lack the abilityfor complex glycosylation (18). Nevertheless, a number ofproteins produced by this system possess biological activity(19-22).

In this paper we report the expression and analysis ofhuman recombinant APP-695, APP-751, and APP-770 pro-duced by insect cells infected with recombinant APP-baculoviruses.

MATERIALS AND METHODSCells and Viruses. Spodopterafrugiperda (Sf9) insect cells

and Autographa californica nuclear polyhedrosis virus (Ac-NPV) were obtained from M. D. Summers (Texas A & MUniversity, College Station) and were cultured in TNMFHmedium (23) containing 10% fetal calf serum.

Cloning of Alternatively Spliced Forms of APP into Bacu-lovirus Expression Vector pJVP10. Full-length clones ofAPP-695, APP-751, and APP-770 were cloned into plasmidpGEM9Zf(-) (Promega). Each of these plasmids was thendigested with restriction endonucleases Xba I and Spe I,yielding a fragment that contained 24 bases of 5' untranslatedsequence, the complete coding region of the APPs followedby 273 residues of the 3' untranslated region. These frag-ments were cloned into the compatible Nhe I restriction siteof vector pJVP10 (from C. Richardson, Biotechnology Re-search Institute, National Research Council ofCanada, Mon-treal), yielding recombinant plasmids termed pJV695,pJV751, and pJV770, respectively.DNA Transfections and Plaque Assays. Plasmids pJV695,

pJV751, and pJV770 were transfected into Sf9 cells togetherwith AcNPV DNA by using the calcium phosphate precipi-tation technique, as modified for insect cells (23). Plaqueassays were done on six-well cluster dishes (GIBCO/BRL)by a modification of the procedure of Vialard et al. (24).

Antibodies. Anti-GID rabbit antiserum was generatedagainst a synthetic peptide (APP 175-187) corresponding tothe NH2-terminal region ofAPP (25). Rabbit affinity-purifiedantibody 369A (from S. Gandy, The Rockefeller University,New York) was raised against a synthetic peptide (APP645-694), corresponding to the COOH-terminal region ofAPP (26). The anti-protease nexin 2 (PN-2) mouse monoclo-nal antibody (mAb) P2-1 has been described (27) and recog-

Abbreviations: APP, amyloid precursor protein; EGF-BP, epidermalgrowth factor-binding protein; AcNPV, Autographa californica nu-clear polyhedrosis virus; mAb, monoclonal antibody; PN-2, proteasenexin 2.

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A K2 3 4 5 6 1 8 9 c10`c 1 314 ja.K 4.Jr "S ~

the^.A.s

OC ~ g. i cat*

7of

K ~~~~~~~4g~~!

4il

p~~~~~~~~~~~~~~~Arl

-~A-

FIG. 1. Immunocytochemical staining of Sf9 cells. Sf9 cells weremock infected (A), infected with AcNPV (B), or infected withrecombinant APP-695-baculovirus (C). Cells were fixed in methanoland stained with mAb P2-1 as described (30). Bound antibody wasdetected with alkaline phosphatase-conjugated secondary antibody(Organon Teknika) and the Immunoselect system (GIBCO/BRL).

nizes an epitope in the NH2-terminal domain of APP(W.E.V.N., unpublished results).Immunoblot Analysis. Infected or mock-infected cells were

pelleted by centrifugation and lysed in electrophoresis sam-ple buffer (24). The harvested medium was concentrated10-fold by using Ultrafree ultrafiltration devices (Millipore).This concentrate was then mixed with an equal volume of 2xelectrophoresis sample buffer. After electrophoresis proteinswere transferred to nitrocellulose or Immobilon poly(vinyl-idene difluoride) membranes (Millipore). The membraneswere then immunoblotted with anti-APP antibodies by one oftwo published procedures (27, 28).

206 5 K -

1109 K - I - S

70 6 K -

43.8K -

B 1 2 3 4 5 6 7 8 9 40 1 1Em 13 '4 1

206 5 K -

110.9 K -

O _

70.6 K -

43.8K -

28 5 K -

FIG. 2. Immunoblot ofintracellular (A) and secreted (B) recombinantAPPs. Sf9 cells were infected with recombinant APP-695 (lanes 1-5),APP-751 (lanes 6-10), and APP-770 (lanes 11-15)-baculovirus, respec-tively. Mediaand cells were harvested 1 day (lanes 1, 6, 11), 2 days (lanes2, 7, 12), 3 days (lanes 3, 8, 13), 4 days (lanes 4, 9, 14), and 5 days (lanes5, 10, 15) after infection. Samples were electrophoresed on SDS/10%oPAGEand immunoblotted with rabbit anti-GID antiserum. Arrows showlocation of recombinant APP proteins. K, kDa.

Purification of Recombinant APPs. Sf9 insect cells wereseeded to =80-90% confluence on 225-cm2 flasks and in-fected with recombinant APP-baculoviruses at a multiplicityof infection of 5. The infected cells were grown in serum-freeExcell 400 medium (JRH Biosciences, Lenexa, KS) at 270Cfor 48 hr. The serum-free medium containing secreted APPproteins was harvested, and protease inhibitor phenylmeth-ylsulfonyl fluoride (0.1 mM) was added. This medium wasthen subjected to low-speed centrifugation (2060 x g for 10min) followed by high-speed centrifugation at 100,000 x g for1 hr in a SW28 rotor. Secreted proteins were then purified bydextran sulfate and affinity chromatography as described (15,27).Amino acid sequence was analyzed at the Facility for

Macromolecular Analysis at State University ofNew York atStony Brook by using an Applied Biosystems model 475Aamino acid analyzer.Protease-APP Complex Formation and Protease Inhibition

Assays. Protease-APP complex formation and protease inhi-bition assays were done as described (9, 15, 27, 29).Growth Restoration of A-1 Fibroblasts. Growth of A-1

fibroblast cells was measured as described (25).

RESULTSExpression of APP-695, APP-751, APP-770 in Sf9 Cells. To

determine that insect Sf9 cells infected with plaque-purifiedrecombinant APP-baculoviruses expressed APP, immunocy-

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tochemical staining with APP-specific antibodies was done.Cells infected with the recombinant APP-baculovirus werestained with NH2-terminal-specific mAb P2-1 (Fig. 1C),whereas mock-infected or wild-type baculovirus-infectedcells showed no staining (Fig. 1 A and B) with this antibody.Similar results were also obtained when recombinant APP-751- or APP-770-infected cells were stained with this anti-body (data not shown). Essentially identical results wereobtained when infected cells were immunostained withCOOH-terminal-specific polyclonal antibody 369A (data notshown). These results show that the recombinant APPs areproduced in infected insect cells and possessed both NH2-and COOH-terminal regions. However, the infected cellsshowed no green birefringence under polarized light afterCongo red staining, indicating the absence of amyloid fibersin these cells (data not shown).Time-course experiments revealed that the recombinant

APPs could be detected in cells and medium 2 days afterinfection (Fig. 2). The levels of these proteins in cells andmedium reached maximum 2 or 3 days after infection (Fig. 2A and B, lanes 2, 3, 7, 8, 12, 13). The estimated molecularmasses ofthe cellular proteins were 116 kDa for APP-695, 120kDa for APP-751, and 122 kDa for APP-770. The estimatedmolecular masses of the secreted proteins were 108.5 kDa forAPP-695 and 110.5 kDa for APP-751 and APP-770. Thedifference in apparent molecular masses between the se-creted and cellular forms suggested that the proteins wereprocessed before secretion. Partial degradation of the pro-teins was evident 3 days after infection (Fig. 2 A and B, lanes3, 8, and 13). The proteins were extensively degraded by thefourth day after infection (Fig. 2 A and B, lanes 4, 9, and 14).No intracellular APP was detected on the fifth day afterinfection (Fig. 2A, lanes 5, 10, and 15).APPs produced in human brain and other tissues are

usually proteolytically cleaved in the middle of the ,/A4domain before secretion (16, 17), resulting in a membrane-associated fragment ofbetween 11 and 16 kDa (26, 31-33). Todetermine whether the recombinant APP proteins expressedin insect cells were similarly cleaved, protein extracts ofinfected insect cells were subjected to electrophoresis in aSDS/15% polyacrylamide gel and immunoblotted withCOOH-terminal-specific antibody 369A (26). A predominantband of '15 kDa was detected with this antibody in cells

1 2 3 4 5 6 7 8200 K -97A K -68 K -

43 K -

29 K -

18.4 K- 34-B]f14.3 K- A

FIG. 3. Immunoblot of Sf9 cells infected with wild-type andrecombinant APP-695, APP-751, and APP-770 baculoviruses. Totalcellular proteins were analyzed at day 2 (lanes 1-4) and day 3 (lanes5-8) after infection. These samples were electrophoresed on aSDS/15% polyacrylamide gel and immunoblotted with affinity-purified rabbit polyclonal antibody 369A. Lanes: 1 and 8, AcNPV-infected cells; 2 and 5, recombinant APP-695 baculovirus-infectedcells; 3 and 6, recombinant APP-751 baculovirus-infected cells; 4 and7, recombinant APP-770 baculovirus-infected cells. Arrow A indi-cates 15-kDa (K) protein, arrow B indicates 19.5-kDa and 20.5-kDaproteins, and arrow C shows 24.5-kDa protein.

1 2 3

200K -116K-97K -

66K -

43K -

FIG. 4. Immunoblot of purified platelet PN-2, human fibroblastPN-2, and recombinant APP-751 secreted from insect cells. Twenty-five nanograms of purified PN-2 from platelets (lane 1), fibroblasts(lane 2), and of purified secreted recombinant APP-751 (lane 3) wasimmunoblotted with mAb P2-1. K, kDa.

infected with the recombinant viruses (Fig. 3, lanes 2-7) butnot in cells infected with wild-type (AcNPV) virus (Fig. 3,lanes 1, 8). Three other proteins with apparent molecularmasses of 19.5 kDa, 20.5 kDa, and 24.5 kDa were alsoobserved. The amount of24.5-kDa protein appeared elevatedon the third day after infection (Fig. 3, lanes 4-7). Productionof the major 15-kDa band suggested that the recombinantAPP molecules expressed in insect cells were cleaved at theCOOH terminal in a fashion analogous to the human APPs.The three other proteins seen may represent intermediates inCOOH-terminal processing of the recombinant APP mole-cules.

Purification and NH2-Terminal Sequence Analysis of Re-combinant APPs. The secreted proteins were purified bydextran sulfate and immunoaffinity chromatography (27).Purified recombinant APP-751 was compared with PN-2purified from fibroblasts or platelets. The purified recombi-nant APP-751 (Fig. 4, lane 3) was seen to comigrate with PN-2purified from platelets (Fig. 4, lane 1) or fibroblasts (Fig. 4,lane 2). Further, purified recombinant APP-695, APP-751,and APP-770 comigrated with PN-2 purified from humanbrain (data not shown).Human APP is usually secreted after cleavage of the first

17 amino acids (3, 15). To determine whether the recombi-nant APPs secreted by insect Sf9 cells were similarlycleaved, purified recombinant APP-695 was subjected toNH2-terminal amino acid sequencing. Although the first twoamino acids could not be determined, the following aminoacids Val-Pro-Thr matched residues 20-22 of APP-695 se-quence (Fig. 5). Additionally, residues 9 and 10 of thesecreted recombinant protein matched those at positions 26and 27 of APP-695. Thus, the recombinant protein secretedby insect cells appears correctly processed at the NH2-terminal signal sequence.

Protease Binding and Inhibition of Protease by RecombinantAPP-751 and APP-770. APP-751 and APP-770, which possessa Kunitz-type protease inhibitor domain, have been shown tobind to and inhibit the activities of some proteases, includingepidermal growth factor-binding protein (EGF-BP), trypsin,

18 19 20 21 22 23 24 25 26 27

Leu Glu Val Pro Thr Asp Gly Asn Ala Gly

X X Val Pro Thr Asp? X Asp? Ala Gly

FIG. 5. Amino acid sequence of secreted recombinant APP-695protein. Numbers indicate positions of amino acids in full-lengthAPP-695 unprocessed protein. Top sequence indicates predictedsequence of secreted protein (15); bottom sequence is that of thepurified secreted recombinant APP-695 protein.

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and chymotrypsin (9, 15, 27, 29). To determine whetherrecombinant APPs possess these activities, purified recom-binant APPs were assayed for complex formation with EGF-BP. Recombinant APP-751 and APP-770 formed stable com-plexes with 125I-labeled EGF-BP (Fig. 6, lanes 2, 3), whereasAPP-695 did not show this binding (Fig. 6, lane 1). Further,the inhibition of trypsin and chymotrypsin activity wasdetermined for each APP spectrophotometrically by moni-toring hydrolysis of a chromogenic substrate with or withoutrecombinant APPs (9, 27, 29). APP-751 and APP-770 inhib-ited the activities of both proteases substantially, whereasAPP-695 did not inhibit protease activity (Fig. 7).Recombinant APP Synthesized by Insect Cells Supports

Growth of Cells That Require APP. The human fibroblast cellline A-1 requires the addition of exogenous APP for completegrowth (25). To test whether the recombinant APPs ex-pressed in insect cells possessed a similar biological activity,media from insect cells infected with wild-type or recombi-nant baculoviruses were tested for the ability to restore cellgrowth of fibroblast cell line A-1. Conditioned medium fromrecombinant APP-695 or APP-751 virus-infected cells in-creased cell growth, whereas conditioned medium from wild-type virus-infected cells had little effect on cell growth (Fig.8). In addition, culture medium from APP-751 virus-infectedcells showed a decrease in ability to support growth at higherconcentrations (Fig. 8).

DISCUSSIONThe baculovirus-insect cell expression system has been usedfor expression of large quantities of recombinant eukaryoticproteins that are usually antigenically and functionally similarto the native proteins. Frequently, these proteins are post-translationally modified and transported in a fashion analo-gous to that of the native protein in eukaryotic cells (18-23).For these reasons this system was chosen as a means ofexpressing cDNAs for APP-695, APP-751, and APP-770. Therecombinant APP proteins expressed in this system peaked inexpression 2-3 days after infection (Fig. 2). The proteinswere secreted, and the signal sequence was appropriatelycleaved.A 11- to 16-kDa APP COOH-terminal fragment has been

observed in rat and human cerebral cortex (31), in APP-695-transfected 293 cells (32), in PC-12 cells (26), and in recom-binant APP-vaccinia virus-infected CV-I monkey fibroblasts(33). This fragment was derived from the full-length protein

80-

60-

40

E 20-

0Control 695 751 770

APP

FIG. 7. Protease inhibition by recombinant APP-695, APP-751,and APP-770. Trypsin or chymotrypsin was incubated alone or withan -5-fold molar excess of purified recombinant APP-695, APP-751,or APP-770. Residual protease activity was measured spectropho-tometrically after hydrolysis of a corresponding chromogenic sub-strate at a wavelength of 405 nm. The percentage of remainingprotease activity after incubation with these recombinant APPs isshown.

by proteolytic cleavage through the 8/A4 domain beforeprotein secretion (16, 17). We found a similar 15-kDa COOH-terminal fragment in insect cells infected with the recombi-nant APP-baculoviruses (Fig. 3, lanes 2-7). The NH2-terminus of this fragment was recently sequenced (34) andfound identical to the NH2-terminus of the APP COOH-terminal fragment identified in mammalian cells (16). Thissimilarity between the COOH-terminal APP fragment ininsect cells and mammalian cells indicates that the recombi-nant baculovirus expression system may provide a goodmodel for characterization of the proteolytic processing ofAPP. We also observed minor COOH-terminal fragments of19.5 kDa, 20.5 kDa, and 24.5 kDa in insect cells infected withthe recombinant APP baculoviruses. These fragments mayrepresent intermediates in the proteolytic processing of re-combinant APP or may represent alternatively processedAPP molecules that contain the entire amyloid ,-proteinregion. Altered proteolytic processing has been hypothesizedas a potential explanation for the accumulation of amyloid,a-protein in individuals with Alzheimer disease (7-9, 35).We found that the secreted recombinant APP-695, APP-

751, and APP-770 were biologically active in two differentassays. Results from the protease-binding studies (Fig. 6)

2

200 K -

116 K -

97 K -

66K -

43K -

3

10,000-

di& 4IE0#4-4 8,000-

6

-uC)

30K - _ _ _

FIG. 6. Autoradiogram of 1251-labeled EGF-BP-recombinantAPP complex formation assays. Approximately 100 ng of purifiedrecombinant APP-695 (lane 1), APP-751 (lane 2), or APP-770 (lane 3)was incubated with -20 ng of 1251-labeled EGF-BP and subjected toSDS/7.5% PAGE with subsequent autoradiography. Bottom bandrepresents uncomplexed 1251-labeled EGF-BP; top band representsthe EGF-BP-APP complex.

6,000 -

4,000-

wAPP-695

Control

0 5Conditioned medium, %

I5

10

FIG. 8. Restoration of growth of fibroblast A-1 cells. The growthassay of fibroblast A-1 cells was done with conditioned medium fromwild-type or recombinant APP-695- or APP-751-baculovirus as de-scribed (25). Numbers of cells are plotted against amount of condi-tioned medium added. Bars show SE.

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show that APP-751 and APP-770 bind to EGF-BP, whereasAPP-695 does not. In addition, APP-751 and APP-770 inhibitactivity of the proteases trypsin and chymotrypsin, whereasAPP-695 does not show this inhibition (Fig. 7). Analogousresults on inhibition of protease activity have been obtainedwith purified PN-2 and from cell extracts of COS-1 cellsexpressing APP-695 or APP-770 (9, 15, 27, 29). The Kunitz-type protease inhibitor domain common to APP-751 andAPP-770 is believed to be involved in the interaction with andbinding to these proteases. APP-695 lacks this domain and,therefore, does not bind to or inhibit these proteases.

Biological activity of APP-695 and APP-751 was furthertested in the growth-regulation assay (25). A fibroblast cellline A-1, transfected with an antisense construct of the 3' endof APP, requires exogenous APP for its full growth, probablybecause endogenous APP production is suppressed by an-tisense RNA for APP. The A-1 cell growth was effectivelyrestored by addition of conditioned medium from recombi-nant APP-695- or APP-751-baculovirus-infected cells (Fig. 8).The higher concentration of recombinant APP-751 did notsupport cell growth, suggesting that APP-751 may havegrowth-inhibitory properties at high concentration. This in-hibitory effect was previously observed with human APP-751overproduced and secreted from mammalian kidney cells(25). Like its native analogue the recombinant APP-695 didnot inhibit growth, even at higher concentrations. This find-ing supports the hypothesis that the physiological functionsof APP-695 and APP-751 are distinct.The baculovirus expression system for APP molecules thus

provides an abundant source of biologically active proteins.These recombinant proteins should help in further charac-terization of the structure, function, and proteolytic process-ing of APP molecules. Furthermore, the role of differentstructural and functional domains in these molecules can bedetermined by studying the effect of systematic deletion ofthese regions.

Note. During the preparation of this manuscript two reports werepublished describing expression of APP-695 (36) and APP-751 (36,37) in insect cells with recombinant baculoviruses.

We thank Dr. M. D. Summers for supplying Sf9 cells and AcNPVDNA, Dr. C. Richardson for plasmid pJVP10 and for technicaladvice, Dr. S. Gandy for antibody 369A, and Dr. M. Vitek for hisadvice and support. This work was supported by Alzheimer'sAssociation Grant IIRG-90-193 to D.G., by National Institutes ofHealth Grant AG00538 to W.E.V.N., and by National Institutes ofHealth Grant AGO5131 to T.S.

1. Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res.Commun. 120, 885-890.

2. Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res.Commun. 122, 1131-1135.

3. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M.,Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther,K. & Muller-Hill, B. (1987) Nature (London) 325, 733-736.

4. Robakis, N. K., Ramakrishna, N., Wolfe, G. & Wisniewski,H. M. (1987) Proc. Natl. Acad. Sci. USA 84, 4190-4194.

5. Goldgaber, D., Lerman, M. I., McBride, 0. W., Saffioti, U. &Gajdusek, D. C. (1987) Science 235, 877-880.

6. Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A. P.,St. George-Hyslop, P. H., Van Keuren, M. L., Patterson, D.,Pagan, S., Kurnit, D. M. & Neve, R. L. (1987) Science 235,880-884.

7. Ponte, P., Gonzales-DeWhitt, P., Schilling, J., Miller, J., Hsu,D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I.,Fuller, F. & Cordell, B. (1988) Nature (London) 331, 525-527.

8. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa-Ko-maroff, L., Gussela, J. F. & Neve, R. L. (1988) Nature (Lon-don) 331, 528-530.

9. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. &Ito, H. (1988) Nature (London) 331, 530-532.

10. deSavage, F. & Octave, J. N. (1989) Science 245, 651-653.

11. Jacobsen, J. S., Muenkel, H. A., Bloom, A. J. & Vitek, M. P.(1991) Neurobiol. Aging 12, in press.

12. Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J. M.,Lemaire, H. G., Kang, J., Muller-Hill, B., Masters, C. L. &Beyreuther, K. (1988) EMBO J. 7, 949-957.

13. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum,J. M., Masters, C. L. & Beyreuther, K. (1989) Cell 57, 115-126.

14. Gandy, S., Czernick, A. J. & Greengard, P. (1988) Proc. Natl.Acad. Sci. USA 85, 6218-6221.

15. Van Nostrand, W. E. & Cunningham, D. D. (1987) J. Biol.Chem. 262, 8508-8514.

16. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A. &Price, D. L. (1990) Science 248, 492-495.

17. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W.,Culwell, A. R., Oltersdorf, T., McClure, D. & Ward, P. J.(1990) Science 248, 1122-1124.

18. Luckow, V. A. & Summers, M. D. (1988) BiolTechnology 6,47-55.

19. Coelingh, L. V. W., Murphy, B. R., Collins, P. L., Lebacq-Verheyden, A. M. & Battey, J. F. (1987) Virology 160, 467-472.

20. Hu, S. L., Kosowski, S. G. & Schaaf, K. F. (1987) J. Virol. 61,3617-3620.

21. Jarvis, D. L. & Summers, M. D. (1989) Mol. Cell. Biol. 9,214-223.

22. Smith, G. E., Ju, G., Ericson, B. L., Moschera, J., Lahm,H.-W., Chizzonite, R. & Summers, M. D. (1985) Proc. Natl.Acad. Sci. USA 82, 8404-8408.

23. Summers, M. D. & Smith, G. E. (1987) A Manual ofMethodsfor Baculovirus Vectors and Insect Cell Procedures (Bulletinno. 1555, Texas Agric. Exp. Stn. and Texas A & M Univ.,University Station, TX).

24. Vialard, J., Lalumiere, M., Vernet, T., Briedis, D., Alkhatib,G., Henning, D., Levin, D. & Richardson, C. (1990) J. Virol.64, 37-50.

25. Saitoh, T., Sundsamo, M., Roch, J.-M., Kimura, M., Cole, G.,Schubert, D., Oltersdorf, T. & Schenk, D. (1989) Cell 58,615-622.

26. Buxbaum, J. D., Gandy, S. E., Cicchetti, P., Ehrlich, M. E.,Czernick, A. J., Fracasso, R. P., Ramabhadran, T. V., Unter-beck, A. J. & Greengard, P. (1990) Proc. Natl. Acad. Sci. USA87, 6003-6006.

27. Van Nostrand, W. E., Wagner, S. L., Farrow, J. S. & Cun-ningham, D. D. (1990) J. Biol. Chem. 265, 9591-9594.

28. Pluskal, M. G., Przekop, M. B., Kavonian, M. R., Vecoli, C.& Hicks, D. A. (1986) Biotechniques 4, 272-282.

29. Van Nostrand, W. E., Wagner, S. L., Suzuki, M., Choi, B. H.,Farrow, J. C., Geddes, J. W., Cotman, C. W. & Cunningham,D. D. (1989) Nature (London) 341, 546-549.

30. Goldgaber, D., Lee, P.-W., Yanagihara, R., Gibbs, C. J. &Gajdusek, D. C. (1985) in Proceedings ofthe FirstInternationalSymposium on Public Health in Asia and the Pacific Basin, eds.Bender, T. R., Diwan, A. R. & Raymond, J. S. (Sch. PublicHlth., University of Hawaii at Manoa, Honolulu), pp. 55-59.

31. Selkoe, D. J., Podlinsky, M. B., Joachim, C. L., Vickers,E. A., Lee, G., Fritz, L. C. & Oltersdorf, T. L. (1988) Proc.Natl. Acad. Sci. USA 85, 7341-7345.

32. Oltersdorf, T. L., Ward, P. J., Henriksson, T., Beattie, E. C.,Neve, R., Lieberburg, I. & Fritz, L. C. (1990) J. Biol. Chem.265,4492-4497.

33. Wolf, D., Quon, D., Wang, Y. & Cordell, B. (1990) EMBO J.9, 2079-2084.

34. Gandy, S. E., Ramabhadran, T. V., Bhasin, R., Koo, E. H.,Price, D. L., Goldgaber, D. & Greengard, P. (1991) J. CellBiol.115, 122a.

35. Abraham, C. R., Selkoe, D. J. & Potter, H. (1988) Cell 52,487-501.

36. Knops, J., Johnson-Wood, K., Schenk, D. B., Sinha, S.,Lieberburg, I. & McConlogue, L. (1991) J. Biol. Chem. 266,7285-7290.

37. Ramakrishna, N., Saikumar, P., Potempska, A., Wisniewski,H. M. & Miller, D. L. (1991) Biochem. Biophys. Res. Com-mun. 174, 983-989.

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