Proc. Nati. Acad. Sci. USAVol. 86, pp. 7731-7735, October 1989Biochemistry

Cell-surface expression and purification of human CD4 produced inbaculovirus-infected insect cells

(human immunodeficiency virus/flow cytometry/epitope mapping/glycosylation/immunoaffinity purification)

NANCY R. WEBB*, CLAUDIE MADOULETt*, PIERRE-FRAN§OIS Tositt, DANA R. BROUSSARD*, LOYD SNEEDtt,CLAUDE NICOLAUtt, AND MAX D. SUMMERS**Department of Entomology and Texas Agricultural Experiment Station, Texas A & M University, College Station, TX 77843; and tBiophor Corporation,Texas A & M University Research Park, College Station, TX 77840

Communicated by Charles J. Arntzen, July 10, 1989

ABSTRACT CD4 is an integral membrane glycoproteinthat acts as the cellular receptor for human immunodeficiencyvirus (HIV). A cDNA encoding full-length CD4 was insertedinto the genome ofAutographa californica nuclear polyhedrosisvirus under transcriptional regulation of the viral polyhedringene promoter. The recombinant virus was used to infect insectcells, which resulted in the abundant expression of CD4 asevaluated by flow cytometry and immunoblot analysis. Recom-binant CD4 expressed on the surface of infected insect cells wasimmunologically indistinguishable from human CD4 whenusing 11 different anti-CD4 monoclonal antibodies. The ex-traction of infected cells by phase-transition separation withTriton X-114 followed by immunoaffinity chromatographyyielded a single protein detected by NaDodSO4/PAGE usingsilver staining. N-terminal sequence analysis of the purifiedrecombinant protein showed that CD4 produced in Sf9 cells isefficiently cleaved from the precursor protein. Immunoblotanalysis under nondenaturing conditions showed that the pu-rified protein reacted with the anti-CD4 monoclonal antibodyLeu-3a. The potential use of the recombinant membrane-associated CD4 in anti-HIV therapy is discussed.

Infection by human immunodeficiency virus (HIV), the caus-ative agent of acquired immune deficiency syndrome (AIDS)(1-3), is mediated by binding to CD4, a glycoprotein ex-pressed on the surface of HIV-susceptible cells (4). Theinteraction between CD4 and the HIV envelope glycoprotein,gpl20, was demonstrated by McDougal et al. (5), whocoimmunoprecipitated the two proteins from infected cells.The affinity constant of the gpl2O-CD4 complex is about 4 x10-9 M, which is comparable to other virus-receptor inter-actions (6). Maddon et al. (7) provided further evidence forthe essential role of CD4 in HIV infection by introducing afunctional CD4 gene into CD4- human cells, thereby con-ferring HIV susceptibility to previously resistant cells.Because of the role of CD4 as the HIV receptor, several

strategies have been proposed for using the CD4 protein toblock infection. Large amounts of a soluble CD4 molecule(sCD4), which contains the extracellular domain but not thehydrophobic transmembrane domain, have been producedby various expression systems. sCD4 binds gp120 and neu-tralizes HIV, as measured by in vitro assays, which includesyncytium induction, virus replication, and target cell cyto-toxicity (8-13). In addition, sCD4 has therapeutic activity invivo (14). sCD4 also has been used as a targeting agent fordelivering cytotoxins to HIV-infected cells that expressgp120 (15).Another potential strategy for blocking HIV infectivity

with CD4 is to insert intact CD4 into the membrane of

long-lived cells not susceptible to HIV. These cells could actas potential scavengers of infected cells expressing the viralenvelope protein gp120 and ofcirculating virus or gp120 in theblood stream (16). We have recently described the stableinsertion of CD4 from human lymphocytes into red bloodcells and showed that extracellular monoclonal antibodybinding epitopes of CD4 were maintained (17, 18). The use ofa CD4-erythrocyte complex as a therapeutic agent for AIDSrequires large amounts of CD4 containing the hydrophobicmembrane-spanning region. We have produced a recombi-nant baculovirus expression vector (19) for the abundantproduction of full-length CD4 in insect cells. We show thatthe recombinant protein expressed on the surface of insectcells is immunologically and biochemically similar to CD4expressed in human cells and describe a method for purifyingthe recombinant product.

MATERIALS AND METHODSCells. Spodoptera frugiperda IPLB-Sf21-AE (20) clonal

isolate 9 (designated Sf9) cells were cultured at 27°C inTNMFH medium (19) supplemented with 10% (vol/vol)heat-inactivated fetal bovine serum (Hazelton/K. C. Biolog-icals, Lenexa, KS) in monolayer or suspension culture (19).A recombinant baculovirus (Ac-CD4) containing a cDNAencoding the full-length CD4 protein under transcriptionalregulation of the polyhedrin promoter was produced bycotransfecting pAc-CD4 DNA (Fig. 1) with wild-type Au-tographa californica nuclear polyhedrosis virus (AcMNPV,strain E2) DNA by calcium phosphate precipitation (23). Theocclusion-negative virus was plaque-purified and propagatedin Sf9 cells (19).Human acute lymphoblastic leukemia cells of the line

CEM-CM3 (American Type Culture Collection) were cul-tured in RPMI 1640 medium supplemented with 10% fetalbovine serum at 37°C in 5% carbon dioxide/95% air.Immunoblot Analysis. Sf9 cells were seeded in six-well

plates at 1 x 106 cells per well and infected with recombinantvirus at a multiplicity of infection of 10. Mock-infected andwild type-infected cells were used as controls. At 24, 36, 48,and 72 hr after infection, cells were lysed in disruption buffer(4% NaDodSO4/0.05 M Tris, pH 6.8/4% 2-mercaptoethanol/10% glycerol/0.05% bromphenol blue), and aliquots repre-senting 4 x 104 cells were applied to a denaturing 10%polyacrylamide gel (24). To estimate the quantity of CD4present in crude lysates, -100 ng of purified recombinant

Abbreviations: HIV, human immunodeficiency virus; sCD4, solubleCD4; AcAMNPV, Autographa californica nuclear polyhedrosis virus;Endo H, endo-f-N-acetyl-D-glucosaminidase H; PE, phycoerythrin.tPresent address: Institute of Biosciences and Technology Center forProtein Engineering, Section of Membrane Biotechnology, TexasA & M University, College Station, TX 77840.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Flanking viralDNA

Flanking viralDNA

EcoRV

FIG. 1. Construction of the plasmid pAc-CD4. DNA manipula-tions were performed by standard procedures (21). The plasmidpT4B (22), which contains the coding sequence of human CD4, wasobtained from R. Axel (Howard Hughes Medical Institute, ColumbiaUniversity). To minimize the amount of CD4 5' flanking sequencesincluded in the recombinant baculovirus expression vector, a Hae IIIcleavage site located 5 base pairs (bp) upstream from the translationinitiation sequence ATG of the CD4 gene was used for cloning. Thepresence of seven additional Hae III sites at the 3' half of CD4required the isolation of the CD4 open reading frame in two separatefragments, as shown. The two fragments were ligated in one step intopBAC-1SH (a gift from Y. Gluzman, Cold Spring Harbor Labora-tory), which contains the EcoRV-BamHI fragment of the polyhedringene (19), where the translation-initiating ATG has been mutated toATT to create a unique Ssp I site. The resulting plasmid (pBAC-CD4)was subsequently digested with EcoRV and Xho II to release a1.6-kilobase (kb) fragment containing the polyhedrin promoter andthe entire coding sequence of CD4. The EcoRV-BamHI fragment ofthe transfer vector pAc311 (19) was replaced with the fragmentderived from pBAC-CD4.

CD4 (see below) was used for comparison. Proteins were

subsequently electroblotted to nitrocellulose membranes byusing a polyblot apparatus (American Bionetics, Emeryville,CA) and probed with a 1:500 dilution of a rabbit anti-CD4anterserum (provided by L. Callahan, Food and Drug Ad-ministration, Bethesda, MD).

Purified CD4 fractions were analyzed -by the method ofTowbin et al. (25) on nondenaturing polyacrylamide gels witha 1:100 dilution of Leu-3a (Becton Dickinson) as the primaryantibody.

Metabolic Labeling and Radioimmunoprecipitation. Sf9cells (1 x 106) were infected with Ac-CD4 virus at a multi-plicity of infection of 10. Mock-infected and wild type-infected cells were used as controls. After a 1-hr absorption

period, the inoculum was replaced with Grace's mediumsupplemented with 0.5% fetal bovine serum. For some ex-periments, 1 ,ug oftunicamycin (Calbiochem Behring) per ml,which efficiently blocks N-glycosylation but not proteinsynthesis in Sf9 cells (26), was also added at this time.Sixteen hours after infection, the medium was removed andreplaced with Grace's medium containing 0.5% fetal bovineserum, 0.5 jig of methionine per ml, and 50 /Ci of '5Stranslabel (ICN) per ml. For treated cells, tunicamycin waspresent throughout the labeling period. At 40 hr after infec-tion, cell extracts were prepared and immunoprecipitationswere performed (26) with 2 1.d of the rabbit antiserum.

Endo-f8-N-acetyl-D-glucosaminidase H (Endo H; Gen-zyme) digestion of radioimmunoprecipitated proteins wasperformed as described (26).

Immunofluorescence Analysis. The following monoclonalantibodies that recognize noncompeting epitopes on the CD4molecule (refs. 27 and 28; P.-F.T., unpublished data) wereused: OKT4A, -B, -C, -D, -E, and -F (provided by P. Rao,Ortho Diagnostics); Mt151 (Boehringer Mannheim); and13B8.2 and BL4/10T4 (AMAC, West Brook, ME). Phyco-eythrin (PE)-conjugated Leu-3a was obtained from BectonDickinson and PE-conjugated goat anti-mouse antibody wasfrom Molecular Probes.For flow cytometry analysis, 5 x 105 cells were incubated

on ice with a saturating concentration of monoclonal anti-body (2 jig), washed, and then stained with the fluorescent-conjugated goat anti-mouse antibody. To avoid the fairlystrong green autofluorescence of Sf9 cells, red fluorescence(PE fluorochrome) was routinely used to follow surface CD4expression. Control cells were stained with either mouseIgG1-PE (Coulter) or with fluorescent conjugate alone.

Cell surface immunofluorescence was quantified by usingan Epics profile (Coulter) and argon laser (488-nm excita-tion). Fluorescence emission was measured by filtering thelight passed by a dichroic mirror through a narrow band filter(575 ± 10 nm). The fluorescence intensity per antibodymolecule (F/P ratio) for the PE-conjugated goat anti-mouseantibody was determined by using Simply Cellular micro-beads, which contain a quantitated number of mouse IgGbinding sites per bead surface (Flow Cytometry StandardsCorporation, Research Triangle Park, NC).

Purification and N-Terminal Sequence Analysis. The isola-tion of membrane-associated CD4 was performed by a mod-ification of the technique of Bordier (29). Infected Sf9 cellswere pelleted by low-speed centrifugation, washed, andresuspended in an extraction buffer containing 0.15 M NaCl,10 mM Tris (pH 8.0), 1% Triton X114, and 0.2 mM phenyl-methylsulfonyl fluoride. After a 20-min incubation on ice, thesample was clarified by centrifugation, and the CD4-containing supernatant was collected. The supernatant wassubsequently incubated at 37°C for 10 min and centrifuged atroom temperature to separate phases. The detergent phasewas harvested, and an equal volume of phosphate-bufferedsaline was added. The resulting mixture was centrifuged 1 hrat 100,000 x g. The supernatant was immediately applied toan immunoaffinity column containing an anti-CD4 monoclo-nal antibody (13B8.2) linked to Affi-Gel 10 beads (4 mg/ml ofgel). The immunoaffinity column was washed with 1 MNaCl/20 mM Tris, pH 7.0, followed by sodium citrate at pH6.0, 5.0, and 4.0. The CD4 fraction was eluted with sodiumcitrate at pH 3.0 and adjusted to pH 7.0.The purified CD4 protein was subjected to N-terminal

sequence analysis (30). The protein was applied to ImmobilonP transmembrane (Millipore) and then covered with trifluo-roacetic acid-treated glass fiber filter. A standard 03RPTHsequencing program was run with a 470A sequencer (AppliedBiosystems). Phenylthiohydantoin-conjugated amino acidswere analyzed on the on-line 120A analyzer. Amino acidanalysis of a purified protein hydrolysate was performed in

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triplicate with the Waters Picotag amino acid analysis system(31). The quantity of purified CD4 was calculated from thetotal amino acid concentration determined by the amino acidanalysis.

RESULTS

Epitope Analysis and Biochemical Properties of Recombi-nant CD4. The expression of membrane-associated CD4 was

detected by immunofluorescent labeling of Sf9 cells infectedwith purified Ac-CD4 virus. Eleven monoclonal antibodies(OKT4A, -B. -C, -D, -E, and -F; MT151; 13B8.2; BL4/1OT4and Leu-3a) were first tested by flow cytometry in cross-

competition studies using CEM-CM3 cells to confirm thateach monoclonal antibody interacts with a distinct extracel-lular epitope of authentic CD4 (data not shown). By using thefluorescence intensity per antibody molecule (F/P ratio)obtained for the PE-conjugated secondary antibody, thenumber of CD4 receptor binding sites on the CEM-CM3 cellsurface was determined to be about 5-6 x 104. Each of themonoclonal antibodies showed similar binding to Ac-CD4-infected Sf9 cells. Indirect fluorescence histogramswere superimposable with a variation of 15%. A typicalhistogram is shown in Fig. 2C. The average ratio of thelinearized red fluorescence measured from the Sf9 cellscompared to that of CEM-CM3 cells was 20, indicating thatAc-CD4-infected Sf9 cells contained -106 antibody bindingsites per cell surface 48 hr after infection.To study the biochemical properties of recombinant CD4,

total cell lysates from Ac-CD4-infected cells were prepared atvarious times after infection, and an aliquot equivalent to 4 X

104 cells was analyzed on immunoblots. An immunoreactiveband with an apparent molecular mass of about 55 kDa thatwas not observed in mock-infected or AcMNPV-infectedcells is present in Ac-CD4-infected cell lysates (Fig. 3A). Thisagrees with the molecular mass of authentic CD4 produced inhuman cells (22). The intensity of immunostaining of the55-kDa band in the crude cell lysates compared with a knownamount of a recombinant CD4 standard (Materials andMethods) indicates that 4 x 104 cells contain at least 100 ngof CD4 48 hr after infection. A direct comparison of theamount of recombinant CD4 produced in Ac-CD4-infectedinsect cells with that expressed by human lymphocyte cellswas not possible because CD4 was not detectable in humancell lysates, even when an extract from 5 x 106 CEM cellswas analyzed (data not shown).

In addition to a major immunoreactive band of -55 kDa,other CD4-related polypeptides were detected in Ac-CD4-infected cells. In Fig. 3A, an immunoreactive band withan apparent molecular mass of 52 kDa was present in the

A

A>Z u

. . L,~ -<

Ba

>7 u-%z

o U , + +

; < Endo TM

-200

-95.5

-55

-43

-36

-97.4

-68

-43

48 24 36 48 72

FIG. 3. Analysis of CD4-related proteins produced in insect cellsby using an anti-CD4 antiserum. (A) Cellular extracts were preparedat the indicated hours after infection (at the bottom ofthe lanes) frommock-infected (lane Mock), wild type-infected (lane AcMNPV), orAc-CD4-infected cells (lanes Ac-CD4), and an aliquot correspondingto 4 x 104 cells was analyzed by NaDodSO4/10% PAGE followed byelectrophoretic transfer to a nitrocellulose membrane. Approxi-mately 100 ng of purified recombinant CD4 is also shown. Molecularweight markers (Diversified Biotech, Newton Centre, MA) areindicated at the right. (B) Radioimmunoprecipitation of recombinantCD4. Sf9 cells mock-infected (lane Mock), AcMNPV-infected (laneAcMNPV), or Ac-CD4-infected (lanes Ac-CD4) were radiolabeledwith 35S translabel in the presence (lane +TM) or absence oftunicamycin. Immunoprecipitates were digested for 0 or 16 hr (lane+Endo) with Endo H. Note that the 98-, 68-, and 60-kDa polypep-tides coimmunoprecipitated from tunicamycin-treated cells (+TM)are not immunologically related to CD4 (see Results). The identity ofthe bands in the Mock and AcMNPV lanes is not known. Since theseproteins are not observed on immunoblots (A), they are not immu-nologically related to CD4.

crude lysates at each time analyzed. In addition, lightlystaining bands with apparent molecular masses of 42 and 39kDa were also detected at 48 and 72 hr after infection.To determine if N-glycosylation might account for the

heterogeneity of CD4 detected on immunoblots, infectedcells were radiolabeled in the presence or absence of tuni-camycin, an inhibitor of asparagine-linked glycosylation (32,33). The anti-CD4 polysera immunoprecipitated two proteins

B C

llJ

z-J

-J

UJ

LOG RED FLUORESCENCE

FIG. 2. Flow cytometry analysis of membrane-bound CD4. Fluorescence histograms of Ac-CD4-infected insect (Sf9) cells incubated withsecondary antibody goat anti-mouse antibody alone (A), human acute lymphoblastic leukemia CEM-CM3 cells incubated with Leu-3a, a

CD4-specific monoclonal antibody that inhibits HIV-induced syncytium formation (28) (B), and Ac-CD4-infected Sf9 cells 48 hr after infectionincubated with Leu-3a. (C). Histograms were generated from 104 cells. Similar histograms were obtained with the following monoclonalantibodies, each of which bind to distinct epitopes on the CD4 molecule (27, 28): OKT4A, -B, -C, -D, -E, and -F; 13B8.2; BL4/10T4 and MT151.No measurable immunofluorescence was detected on AcMNPV or on mock-infected Sf9 cells.

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from untreated cells radiolabeled 16-40 hr after Ac-CD4infection, corresponding to the 55- and 52-kDa forms de-tected by immunoblot analysis (Fig. 3A). Radioimmunopre-cipitation of tunicamycin-treated Ac-CD4-infected cellsyielded several major bands (Fig. 3B, lane +TM). However,only the band migrating with an apparent molecular mass ofabout 52 kDa was specific, as revealed by immunoblotanalysis (data not shown). Since the other proteins observedin the immunoprecipitation experiment (apparent molecularmasses of 98, 68, and 60 kDa) were not detected by immu-noblot, they may be proteins of viral or cellular origin thatnonspecifically coprecipitate as a result of a physical asso-ciation with the CD4-antibody complex. Proteins of un-known source or function have been shown to coimmuno-precipitate with the nonglycosylated forms of tissue plasmi-nogen activator and other foreign proteins expressed by arecombinant baculovirus in tunicamycin-treated Sf9 cells(ref. 26; D. Jarvis, C. Oker-Blom, and M.D.S., unpublisheddata). Endo H digestion ofradioimmunoprecipitated proteinsfrom Ac-CD4-infected Sf9 cells yielded a single CD4 band,which comigrated with the specific band observed in tuni-camycin-treated cells (Fig. 3B, lanes +Endo and +TM). Thisresult supports the conclusion that the higher molecular massform ofCD4 is N-glycosylated and that Sf9 cells infected withAc-CD4 contain both glycosylated and nonglycosylatedforms of CD4. The identity of the 42- and 39-kDa polypep-tides observed at 48 and 72 hr after infection (Fig. 3A) is notknown. These additional immunoreactive peptides were notconsistently observed for all immunoblot experiments, and itmay be that they represent proteolytic products of CD4.

Purification and N-Terminal Sequence Analysis of Recom-binant CD4. The use of Triton X-114 to solubilize membraneproteins of Ac-CD4-infected Sf9 cells, followed by a tem-perature-induced separation of aqueous and detergentphases, resulted in the recovery of 20% of the membrane-associated CD4 in the detergent phase. The solubilized CD4was further purified by immunoaffinity chromatography withthe anti-CD4 monoclonal antibody 13B8.2. The pH 3.0 eluantfrom this column was analyzed by NaDod S04/PAGE, anda single protein with an apparent molecular mass of about 55kDa was detected by silver staining (Fig. 4A). Immunoblotanalysis under nondenaturing conditions showed that theeluted protein reacted with the anti-CD4 monoclonal anti-body Leu-3A (Fig. 4B).The N-terminal 14 amino acids of the affinity-purified

recombinant CD4 was determined to be Xaa-Xaa-Val-Val-Leu-Gly-Lys-Lys-Gly-Asp-Xaa-Val-Glu-Leu. Althoughthe identity of the first two amino acids was not verified, the

A1 2

130-

75 -

50 -

39

27

17 -

B1 2

130 -

75 -

-0-

39 -

27 -

FIG. 4. Gel electrophoresis (A) and immunoblot analysis (B) ofpurified recombinant CD4. CD4 was extracted and purified asdescribed. An aliquot from the 100,000 x g supernatant of theinfected cell extract (lanes 1) or the pH 3.0 eluant from an immu-noaffinity column (lanes 2) was detected by silver staining onNaDodSO4/10% PAGE (A) and by immunoreactivity with Leu-3amonoclonal antibody under nondenaturing conditions (B).

positioning of the amino acids for the signal sequence cleav-age site of the processed CD4 is unambiguous. The aminoacid sequence determined from the insect-derived CD4 alignswith amino acids 26-39 predicted from the nucleotide se-quence ofthe CD4 cDNA (22). This unambiguously identifiesthe signal sequence cleavage site after amino acid 25 of theCD4 precursor protein. This result agrees with the cleavagesite determined previously for soluble CD4 expressed inChinese hamster ovary cells (11) and in insect cells (12).

DISCUSSIONA variety of prokaryotic and eukaryotic genes have beenexpressed in S. frugiperda insect cells infected with a re-combinant AcAMNPV. The recombinant proteins produced inthe baculovirus system are functionally, antigenically, andbiochemically similar to their authentic counterparts (34).The results presented here demonstrate the cell-surfaceexpression of CD4 in Sf9 cells infected with a recombinantbaculovirus, Ac-CD4, which contains a cDNA encoding thefull-length CD4 protein. N-terminal sequence analysis of therecombinant protein showed that CD4 produced in Sf9 cellsis efficiently cleaved from the precursor protein. A total of 11different anti-CD4 monoclonal antibodies, shown by crosscompetition studies to interact with distinct CD4 epitopesexpressed on the CEM-CM3 cell surface, were able to bindto the surface of Ac-CD4-infected Sf9 cells. The nature ofbinding of each monoclonal antibody to the Ac-CD4-infectedcells indicates that the tertiary structure ofrecombinant CD4is similar to authentic CD4. The fact that CD4 expressed onthe surface of insect cells binds monoclonal antibodies thatare known to block HIV infection (OKT4A, OKT4F, Leu-3a,and MT151) (27, 28) indicates that the recombinant CD4could serve as a receptor for HIV gpl20. Preliminary resultstesting the ability of recombinant CD4 to bind to gpl20support this conclusion (unpublished data).Radioimmunoprecipitation studies and immunoblot analy-

sis showed the presence of two CD4-related polypeptides inAc-CD4-infected cells (apparent molecular masses of 55 and52 kDa). The 52-kDa polypeptide comigrated with nonglyco-sylated CD4 expressed in tunicamycin-treated cells. Endo Hdigestion of the recombinant CD4 generated a product thatalso comigrated with the CD4 detected in tunicamycin-treated Sf9 cells, showing that CD4 is N-glycosylated in Sf9cells. The sensitivity of the 55-kDa CD4 to Endo H, which iscapable of removing only high-mannose sugar residues (35),suggests that the N-linked oligosaccharides of the glycosyl-ated CD4 are not processed to a complex form. The nature ofglycosylation of the recombinant CD4 protein is similar tomany other mammalian glycoproteins expressed in Sf9 cells,where conversion to an Endo H-resistant form is not detected(36-39).

Analysis of the amino acid sequence predicted by the CD4cDNA reveals two potential N-linked glycosylation sites (22).A study by Konig et al. (40) shows that tunicamycin treat-ment ofCEM cells decreases the apparent molecular mass ofCD4 from 52 kDa to 46 kDa, which is consistent withglycosylation at both putative N-linked sites. Since N-terminal sequence analysis of purified recombinant CD4showed that the signal peptide was efficiently cleaved fromthe CD4 precursor expressed in insect cells, we assume thatthe nonglycosylated CD4 observed in tunicamycin-treatedinsect cells (apparent molecular mass of 52 kDa) is similar, ifnot identical, to the nonglycosylated CD4 reported for tuni-camycin-treated human cells (apparent molecular mass of 46kDa). Direct chemical analysis of the oligosaccharide moi-eties present on authentic and recombinant CD4 has not beendone.The presence of significant amounts of the nonglycosylated

CD4 precursor in Ac-CD4-infected insect cells differs from

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the results described for CD4 expressed in human cells,where nonglycosylated CD4 is not detected on immunoblots(40). This difference may be due to the relative inefficiencyof processing ofCD4 in insect cells compared with the humancells or the relative stability ofthe nonglycosylated protein inthe two cell lines. The fact that an accumulation of nongly-cosylated CD4 is not detectable by immunofluorescencemicroscopy of tunicamycin-treated CEM cells suggests thatthe nonglycosylated CD4 may be labile in these cells (40).The level of expression of the recombinant protein was

compared to the level expressed in human lymphocytes bytwo methods. Flow cytometry analysis with 11 differentCD4-specific monoclonal antibodies indicated that the cell-surface expression of recombinant CD4 is -20-fold higherthan that measured on human CEM-CM3 cells. Immunoblotanalysis of total cell extracts suggested that the expression ofCD4 in infected Sf9 cells is at least 100-fold higher than inCEM cells. The discrepancy between the two assays may bedue to the inherent sensitivity of each quantitation methodused or the method by which each was standardized. Alter-natively, it is possible that Ac-CD4-infected Sf9 cells containa significant amount of CD4 not expressed on the cellmembrane.The extraction and purification methods described here

yielded a highly purified membrane-derived CD4 with amolecular mass of 55 kDa and immunoreactivity to Leu-3amonoclonal CD4 antibody. Insertion of recombinant CD4molecules into red blood cell membranes or reconstitution inliposome membranes (16, 18) may yield CD4 carriers capableof interaction with gpl20-expressing cells, HIV, or gp120antigen, and possibly, in the case of red blood cells, along-lived CD4 carrier.

We are indebted to R. Axel for donating the CD4 cDNA, L.Callahan for the rabbit anti-CD4 polyserum, and Y. Gluzman forpBAC-1SH. We thank Dr. Timothy Hayes for N-terminal sequenceanalysis and Don Jarvis, Christian Oker-Blom, Bruce Webb, andLinda Guarino for helpful discussion. The excellent technical assist-ance of Lee Park and Jeff Spaw is gratefully acknowledged. Thisresearch was funded in part by a contract from Biophor Corporation,the Texas Advanced Technology Research Program, and TexasAgricultural Experiment Station Project TEX06316.

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