recombinant protein synthesis in chinese hamster ovary cells

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  • Recombinant Protein Synthesis in Chinese HamsterOvary Cells Using a Vaccinia Virus/Bacteriophage T7Hybrid Expression System*

    (Received for publication, February 13, 1996, and in revised form, April 24, 1996)

    Anna Ramsey-Ewing and Bernard Moss

    From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892

    The vaccinia virus/bacteriophage T7 expression sys-tem was adapted to Chinese hamster ovary (CHO) cells.Vaccinia virus undergoes abortive infection in CHOcells, which is characterized by a sharp reduction inprotein synthesis at the stage of viral intermediate geneexpression. We determined that expression of a T7 pro-moter-regulated chloramphenicol acetyltransferasegene was at least 20 times more efficient in permissiveBS-C-1 than in CHO cells. The encephalomyocarditis vi-rus 5*-untranslated region, which confers cap-indepen-dent translatability to mRNA, stimulated recombinantprotein synthesis by 10-fold in both cell lines, maintain-ing the advantage of the BS-C-1 cells over CHO cells.Since the cowpox virus hr gene overcomes vaccinia vi-rus host range restriction in CHO cells, we constructeda recombinant virus that carries an intact hr gene inaddition to the T7 RNA polymerase gene. With this virus,synthesis of T7 RNA polymerase was enhanced and pro-duction of a recombinant protein occurred in CHO cellsat the level observed in permissive cell lines. Extensionof the vaccinia virus/bacteriophage T7 expression sys-tem to CHO cells should be of wide interest, as thesecells have advantages for preparation of recombinantproteins in research and biotechnology.

    Recombinant expression systems may use regulatory ele-ments derived from phylogenetically distinct lineages. A suc-cessful mammalian expression system that exploits the bacte-riophage T7 RNA polymerase to transcribe T7 promoter-regulated target genes was described using vaccinia virus(VV)1 as the vector (1). In this system, recombinant VV carriesan integrated T7 polymerase gene regulated by a viral pro-moter. Originally, the T7 promoter-regulated template wascarried on either a plasmid that was transfected into infectedcells or on a second recombinant virus that was coinfected withthe T7 polymerase-expressing virus (2, 3). The low translat-ability of the largely uncapped mRNAs was overcome by initi-ating T7 transcripts with the leader sequence of encephalomyo-carditis virus (EMCV) (4). More recently, the VV/T7 hybridsystem was rendered inducible by inclusion of the Escherichiacoli lac operator/repressor system (5, 6), thereby allowing the

    construction of recombinant viruses containing both the T7RNA polymerase and the T7 promoter-regulated gene. Becausevaccinia virus infects a broad range of cells in tissue culture,this system has been used to express recombinant proteins in avariety of cells.One notable exception to the usable cell lines is the Chinese

    hamster ovary (CHO) cell line. Despite the fact that CHO cellsare one of a few cell lines approved for production of recombi-nant proteins for use in clinical trials, the VV/T7 technologyhas not yet been adapted to these cells. One major obstacle hasbeen the restriction in growth and gene expression of VV inCHO cells (7). Insertion of the CHO hr gene of cowpox virus(CPV) into the genome of VV, however, enables VV to produc-tively infect CHO cells (8). Replication of VV on CHO cells isblocked at the stage of viral intermediate protein synthesis (9).This defect is overcome by the CHO hr gene (9), which alsodelays the onset of apoptosis in VV-infected CHO cells (10). Weconstructed a new recombinant VV containing both the CHO hrand the bacteriophage T7 RNA polymerase genes, which allowscomparable levels of expression in CHO and fully permissivecell lines. Expression was further enhanced by incorporatingthe EMCV untranslated leader sequence.

    MATERIALS AND METHODS

    Cells and VirusesBS-C-1 (kidney, African green monkey) and CV-1(kidney, African green monkey) cells were grown in minimum essentialmedium supplemented with 5% fetal calf serum. HeLa S3 monolayercultures were maintained in Dulbeccos modified Eagles medium sup-plemented with 10% fetal calf serum. CHO cells were grown in mono-layer culture in Hams F-12 medium supplemented with 5% fetal calfserum. In suspension culture, CHO cells were maintained in an equalmixture of Hams F-12 and CHO-S-SFM II (Life Technologies, Inc.) orUltraCHO (BioWhittaker) media. VV strain WR and recombinantsthereof were propagated as described (11).Construction of Recombinant VirusesRecombinant viruses vTF73,

    vT7EMCAT, and vT7CAT have been described elsewhere (2, 3) and aredepicted in Fig. 1. To construct the vT7CP recombinant virus, a 2.3-kilobase pair EcoRI/PstI fragment containing the entire open readingframe of the CPV hr gene CP77 was excised from pEA36 (8) and ligatedto EcoRI/PstI cleaved pUC19. The resultant plasmid, pRECP77 (9), wasused as a donor in homologous recombination with vTF73 to createvT7CP. In addition to the T7 polymerase gene regulated by a viralearly/late promoter (P7.5) at the thymidine kinase locus, recombinantvirus vT7CP has an intact copy of the CP77 open reading frame, insteadof the disrupted WR copy (12), at the corresponding region ofHindIII C.Analysis of Viral Protein SynthesisTo examine viral protein syn-

    thesis, we seeded 5 3 105 permissive or nonpermissive cells in mini-mum essential medium with 5% fetal calf serum. After 1624 h, thecells were coinfected with recombinant viruses at a total multiplicity of30 plaque-forming units (pfu)/cell, 15 pfu/cell for each virus. 1020 minbefore each labeling period, cells were washed twice and incubated inminimum essential medium with 5% fetal calf serum without methio-nine. The cells were then incubated in the presence of 75 mCi of [35S]me-thionine in 250 ml of methionine-free minimum essential medium with5% fetal calf serum for 30 min. The labeling medium was removed andthe cells were washed twice with ice-cold phosphate-buffered saline(PBS) and then incubated at 37 C for 35 min with hypotonic lysis

    * The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact. To whom correspondence should be addressed: Bldg. 4, Rm. 229,

    NIAID, National Institutes of Health, Bethesda, MD 20892. Tel: 301-496-9869; Fax: 301-480-1147; E-mail: bmoss@nih.gov.

    1 The abbreviations used are: VV, vaccinia virus; CPV, cowpox virus,EMCV, encephalomyocarditis virus; CHO, Chinese hamster ovary; hr,host range; hpi, h postinfection; PBS, phosphate-buffered saline; SDS,sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; pfu,plaque-forming unit; CAT, chloramphenicol acetyltransferase.

    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 28, Issue of July 12, pp. 1696216966, 1996Printed in U.S.A.

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  • buffer (20 mM Tris-HCl (pH 8.0), 10 mM NaCl, and 0.5% Nonidet P-40).The lysates were collected and centrifuged for 2 min at 12,000 3 g tosediment nuclei. The supernatants containing 35S-labeled polypeptideswere stored at 220 C. A portion of each sample was mixed with anappropriate volume of 3 3 or 5 3 sodium dodecyl sulfate/b-mercapto-ethanol (SDS/2-mercaptoethanol, 59 to 39, Inc.) sample buffer and boiledfor 5 min. The proteins were resolved by polyacrylamide gel electro-phoresis (PAGE) in 10, 12, or 15% gels.ImmunoprecipitationCells were infected with 15 pfu/cell of each

    recombinant virus, labeled with [35S]methionine as described above,and harvested at various times. Lysates were prepared in isotonic lysisbuffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide,0.1% SDS, 100 mg/ml phenylmethylsulfonyl fluoride, and 1% NonidetP-40), centrifuged to remove nuclei, and then incubated with polyclonalantiserum to CAT at a 1:500 dilution at 4 C overnight. An equalvolume of 20% (w/v) protein A-Sepharose beads in PBS was added andincubation continued at room temperature for an additional 23 h or at4 C overnight. Immune complexes were washed twice in Triton buffer(300 mM NaCl, 50 mM Tris-HCl (pH 7.4), and 0.1% Triton X-100),denatured and solubilized in Laemmli buffer, and then resolved bySDS-PAGE in 15% gels.Western Blot AnalysisFor analysis of steady-state levels of pro-

    teins, Western blot analysis was performed. Infected cells (1 3 106)were incubated with 0.5 ml of lysis buffer (20 mM Tris-HCl (pH 7.0),0.5% Triton X-100 in PBS) for 5 min at 37 C. Lysates were centrifugedat 14,000 3 g for 5 min and the supernatants stored at 220 C. Aportion was mixed with 5 3 SDS/2-mercaptoethanol sample buffer andboiled for 5 min, and proteins were resolved by SDS-PAGE in 10% gels.Proteins were electrophoretically transferred to nitrocellulose mem-branes and incubated with antisera (anti-CAT 1:500 and anti-T7 po-lymerase 1:1000) overnight at 4 C and then with 125I-protein A over-night at 4 C.Preparation and Analysis of Infected Cell RNARNA was isolated

    from cells that had been infected with two recombinant viruses at amultiplicity of 15 pfu/cell for each. At various times after infection, thecells were washed twice in ice-cold PBS, and then they were lysed andthe RNA was extracted by the guanidinium thiocyanate/phenol/chloro-form extraction method (13), using RNAzol B, as described by themanufacturer (Tel-Test).To quantitate the steady-state levels of RNAs, samples of infected

    cell RNA were applied to a nylon membrane (Schleicher & Schullnytran) using a Hoefer 24-well slot blot apparatus. Membranes werehybridized with a 32P-labeled DNA probe complementary to CATmRNA.Analysis of Reporter Gene ExpressionCells were infected with re-

    combinant viruses at a multiplicity of 30 pfu/cell. At various

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