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WO1996002563A9 - Proteine de l'antigene nucleaire 1 du virus epstein barr, son expression et sa recuperation - Google Patents

Proteine de l'antigene nucleaire 1 du virus epstein barr, son expression et sa recuperation

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WO1996002563A9
WO1996002563A9 PCT/US1995/008700 US9508700W WO9602563A9 WO 1996002563 A9 WO1996002563 A9 WO 1996002563A9 US 9508700 W US9508700 W US 9508700W WO 9602563 A9 WO9602563 A9 WO 9602563A9
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polypeptide
protein
ebnal
gly
ebnal protein
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  • the present invention relates to Epstein-Barr virus nuclear antigen 1 (EBNAl) protein and its expression and recovery. More particularly, the present invention relates to a process for recovering EBNAl protein or polypeptide from cells having a nucleus containing expressed EBNAl protein or polypeptide.
  • EBNAl Epstein-Barr virus nuclear antigen 1
  • Epstein-Barr virus a human herpesvirus, is one of the most common viruses infecting man, and antibodies to EBV proteins are present in greater than 80% of human serum samples. Milman et al . , "Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man," Proc. Natl. Acad. Sci. USA, 82:6300-04 (1985) , which is hereby incorporated by reference.
  • Epstein and Barr reported the first successful attempt to establish continuous lymphoblastoid cell lines from explants of Burkitt's lymphoma ("BL”), which were eventually found to be infected with EBV by W. and G. Henle in 1966. Epstein et al . , "Cultivation in vi tro of human ly ⁇ nphoblasts from Burkitt's malignant lymphoma," Lancet. 1:252-53 (1964) and Henle et al . , "Immunofluorescence in cells derived from Burkitt' s lymphoma," J. Bacteriol . , 91:1248-1256 (1966) , which are hereby incorporated by reference.
  • EBV In addition to its involvement in BL, EBV is the etiological agent of infectious mononucleosis and has been implicated in the pathogenesis of nasopharyngeal carcinoma. EBV can also induce fatal lymphoproliterative disease, sometimes with the features of frank lymphoma, in certain patients with global immunodeficiency that is either congenital (such as severe combined immunodeficiency or ataxia telangiectasia) or acquired as the result of immunosuppression for organ or tissue transplantation or due to AIDS. Kieff et al . , "Epstein-Barr Virus and Its Replication, " Chapter 67, pp.
  • EBV nuclear antigen 1 EBV nuclear antigen 1
  • the 172, 000-base-pair (“bp") DNA genome of EBV is found in all "immortalized” permanent B-cell lymphoblast lines as multicopy latent extrachromosomal circular DNA plasmids or episomes.
  • Only EBNAl is essential for the replication of these EBV plasmids.
  • the EBNAl protein comprises 641 amino acids ("aa”) .
  • One-third of EBNAl (aa 90 to 325) consists of a repetitive array of glycine ("Gly”) and alanine (“Ala”) amino acid residues.
  • Gly- Ala repeat sequence has homology to cellular DNA, and antisera to Gly-Ala repeat-containing peptides also react with cellular proteins, e.g., E. coli , mammalian or baculovirus cellular proteins containing glycine plus alanine-rich regions. Id.
  • EBNAl protein binds in trans to the latent origin of replication, oriP, at multiple sites present in the two regions of oriP which were found to be necessary and sufficient for origin function.
  • One of these regions is composed of 20 tandem copies of a 30-bp sequence (i.e., family of repeats) , each of which contains an EBNAl binding site.
  • the other region includes four EBNAl binding sites (dyad symmetry element) , two of which are located within a 65-bp region of dyad symmetry.
  • the interaction of EBNAl with oriP occurs mainly through the carboxyl-terminal third of the protein.
  • EBNAl activates oriP to function not only as an origin of replication but also as a plasmid maintenance element and a transcriptional enhancer. Frappier et al . , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus," The Journal of Biological Chemistry. 266 (12) :7819-26, (1991) , and Yates et al. , "Dissection of DNA Replication and Enhancer Activation Functions of Epstein-Barr Virus Nuclear Antigen 1," Cancer Cells 6/Eukaryotic DNA Replication, pp. 197-205, Cold Spring Harbor Laboratory, 1988, which are hereby incorporated by reference .
  • EBNAl Can Link the Enhancer Element to the Initiator Element of the Epstein-Barr Virus Plasmid Origin of DNA Replication, " Journal of Virology. 66(l) :489-95 (1992) , which is hereby incorporated by reference, expressed EBNAl in CV-lp cells by using an infectious simian virus (SV) 40 vector containing the EBNAl gene. Expression was quite poor.
  • SV infectious simian virus
  • One aspect of the present invention relates to a process for recovering EBNAl protein or polypeptide.
  • cells having a nucleus containing expressed EBNAl protein or polypeptide are treated to recover the nucleus containing the expressed EBNAl protein or polypeptide.
  • the nucleus containing the expressed EBNAl protein or polypeptide is then separated into a liquid fraction containing the expressed EBNAl protein or polypeptide and a solid fraction containing substantially all DNA from the nucleus.
  • the liquid fraction is separated from the solid fraction, and EBNAl protein or polypeptide is recovered from the liquid fraction.
  • This process produces abundant quantities of purified EBNAl protein or polypeptide useful for diagnosis of EBV.
  • the present invention also relates to an isolated
  • This isolated EBNAl protein or polypeptide formulation having substantially no components which generate false positive readings when used to detect EBV in human serum.
  • This isolated EBNAl protein or polypeptide formulation can be utilized for detection of EBV in a sample of human tissue or body fluids. This detection process involves providing the isolated EBNAl protein or polypeptide formulation as an antigen, contacting the sample with the antigen, and detecting any reaction which indicates that EBV is present in the sample using an assay system.
  • the present invention provides an isolated DNA molecule encoding EBNAl protein or polypeptide, a recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA molecule encoding EBNAl protein or polypeptide, and a host cell incorporating a heterologous DNA molecule encoding EBNAl protein or polypeptide, all of which have substantially no components which generate false positive readings when used to detect EBV in human serum.
  • the present invention also provides a process of expressing an EBNAl protein coding sequence in a cell. In this process, an EBNAl protein coding sequence is cloned into a baculovirus transfer vector.
  • the baculovirus transfer vector and Autographica calif ornica nuclear poly edrosis genomic DNA are then co-transfected into insect cells, and recombinant baculoviruses are recovered. Cells are then infected with the recombinant baculovirus under conditions facilitating expression of isolated EBNAl protein or polypeptide in the cell.
  • the EBNAl protein coding sequence includes no more than 90% of the
  • Gly-Ala repeat amino acid sequence present in the naturally- occurring EBNAl protein coding sequence which spans the Gly- Ala repeat amino acid sequence.
  • EBNAl protein or polypeptide is expressed in quantities sufficient for the production of a detection immunoassay for EBV which provides few false positive readings.
  • FIG. 1 shows the construction of the EBNAl baculovirus transfer vector pVL941-EBNAl.
  • the sequence of the oligonucleotide linkers inserted in the polyhedrin gene of the baculovirus transfer vector pVL941-SW is shown above the plasmid.
  • the underlined ATG is the only ATG sequence in the 5' region of the polyhedrin gene and was used as the start codon for translation of the EBNAl gene.
  • the 3 '-recessed ends were extended with the Klenow fragment of D ⁇ A polymerase I.
  • the EBNAl gene was excised from p205 with Rsal and Ball enzymes, which remove the first seven codons of the gene, and ligated into pVL941-SW to form pVL941-EBNAl .
  • Hygromycin phosphotransferase (hph) and /3-lactamase ⁇ amp) genes are also shown.
  • FIG. 2 shows a modified protocol for improved yield and purity of bEBNAl .
  • This is a Coomassie Blue stained SDS-polyacrylamide gel analysis of each step in the new purification scheme. The lanes read from right to left instead of from left to right. Starting at the far right the lane marked "EBNAl" is a lane of bEBNAl protein purified by this procedure (i.e. it is the same as the lane on the far left) as verified by ability to bind to oriP. Cells - are whole SF-9 cells infected with the recombinant bEBNAl recombinant baculovirus and the bEBNAl band is visible.
  • Cytoplasm - is the cytoplasmic supernatant after lysing the cells and spinning down the nuclei.
  • Nuclei - is the whole nuclei after cell lysis and separating out nuclei from cytoplasm by centrifugation.
  • PolyminP - is the supernatant after lysis of the nuclei and pelleting the DNA by PolyminP and centrifugation.
  • 30% A.S. - is the pellet that forms upon adding ammonium sulfate to the PolyminP supernatant (no significant bEBNAl present) .
  • 45% A.S. is the pellet that forms upon adding ammonium sulfate to the 30% A.S.
  • FIG. 3 shows the phosphate labeling and phosphatase digestion of bEBNAl.
  • Sf-9 cells were infected with the AcMNPV-EBNAl baculovirus and labeled with [ 32 P] orthophosphate as described in the Examples. Labeled cells were separated into cytoplasmic (cyt) and nuclear ( ⁇ uc) fractions, and bEBNAl was purified to homogeneity from the nuclear extract. Pure [ 32 P] EBNAl was incubated at 25°C for 1 h either with (+) or without (-) CIP. Samples were subjected to electrophoresis on 12% SDS-polyacrylamide gels and 32 P-labeled proteins were detected upon autoradiography of wet gels.
  • FIG. 3 shows the phosphate labeling and phosphatase digestion of bEBNAl.
  • FIGS. 5A and B show the native aggregation state of bEBNAl.
  • bEBNAl was combined with the protein standards apoferritin (apo; 440 kDa) , IgG (158 kDa) , bovine serum albumin (BSA; 66 kDa) , ovalbumin ( ova; 45 kDa) and myoglobin ( yo; 17 kDa) , then analyzed by glycerol gradient sedimentation (A) or gel filtration on Superose (B) as described herein.
  • bEBNAl was identified in column fractions by the nitrocellulose filter binding assay. The sedimentation coefficient (s) and Stokes radius of bEBNAl were determined by comparison to the positions of protein standards of which the s values and Stokes radii are known.
  • FIG. 6 shows the stoichiometry of [ 35 S]bEBNAl bound to oriP DNA.
  • [ 35 S] bEBNAl was incubated with pGEMoriP7, then gel-filtered to separate [ 35 S]bEBNAl bound to pGEMoriP7 in the excluded fractions from unbound bEBNAl in the included fractions as described in the Examples. Fractions were analyzed for DNA and [ 35 S] bEBNAl.
  • FIG. 7 shows the salt dependence of bEBNAl binding to the family of repeats and the dyad symmetry element.
  • bEBNAl 50 ng was incubated with 40 fmol of 32 P-end-labeled DNA containing either the dyad symmetry element ( closed circles) or the family of repeats (open circles) in the presence of 2.5 ⁇ g of calf thymus DNA and various concentrations of NaCl. After 10 min at 23°C, the reaction mixture was filtered through nitrocellulose, and the DNA retained on the filters was quantitated by liquid scintillation.
  • FIG. 8 shows the effect of the family of repeats on binding of bEBNAl to the dyad symmetry element .
  • Top diagram of oriP showing the disposition of EBNAl binding sites (boxes) .
  • Bottom 10 fmol of 32 P-labeled DNA fragment containing either the family of repeats ( open circles) , the dyad symmetry element ( closed circles) , or the complete oriP ( closed triangles) were incubated with various amounts of bEBNAl (shown as fmol dimers) in 50 mM HEPES (pH 7.5) , 300 mM NaCl, 5 mM MgCl 2 for 10 min at 23°C.
  • Reactions containing the family of repeats or dyad symmetry element were then filtered through nitrocellulose.
  • Reactions containing the complete oriP closed triangles
  • Reactions containing the complete oriP were treated with 50 units of EcoRV for 3 min at 37°C to separate the family of repeats from the end-labeled dyad symmetry element (see scheme, top) prior to filtration through nitrocellulose.
  • FIGS. 9A and B show the protection of the Aval site in the dyad symmetry element by bEBNAl.
  • FIG. 9A the 300-bp DNA fragment containing the dyad symmetry element, 32 P-end-labeled at one end only, was incubated with various amounts of bEBNAl (shown as fmol dimers) prior to digestion with Aval and electrophoresis on a 6% polyacrylamide gel. The DNA was visualized by autoradiography of dried gels.
  • Scheme of DNA fragment (top) shows EBNAl consensus binding sites (boxes) .
  • FIG. 9A the 300-bp DNA fragment containing the dyad symmetry element, 32 P-end-labeled at one end only, was incubated with various amounts of bEBNAl (shown as fmol dimers) prior to digestion with Aval and electrophoresis on a 6% polyacrylamide gel. The DNA was visualized by autoradiography of dried gels.
  • FIG. 10 is cloning scheme for preparation of a vector for expression in E. coli of EBNAl.
  • FIG. 11 is a map for the plasmid p291.
  • the HindiII fragment contains the eEBNAl gene's nucleotides 107930-110493 (2.563kb) from the strain EBV B93-8, with the eEBNAl gene itself spanning nucleotides 107950 to 109872
  • FIGS. 12A-C show the full double stranded DNA PCR product of the eEBNAl gene with restriction endonuclease sites.
  • the upper strand corresponds to SEQ. ID. No. 3.
  • the present invention relates to a process for recovering EBNAl protein or polypeptide having the following steps: providing cells having a nucleus containing EBNAl protein or polypeptide; recovering the nucleus containing expressed EBNAl protein or polypeptide from the cells; separating the nucleus containing expressed EBNAl protein or polypeptide into a liquid fraction containing the expressed EBNAl protein or polypeptide and a solid fraction containing substantially all DNA from the nucleus; separating the liquid fraction from the solid fraction; and recovering EBNAl protein or polypeptide from the liquid fraction.
  • the nucleus is separated by centrifugation where the liquid fraction is a supernatant and the solid fraction is a pellet. After centrifugation, the supernatant contains less than 5% of DNA.
  • the process further provides subjecting the liquid fraction to a first ammonium sulfate treatment at an ammonium sulfate concentration which forms a solid phase containing contaminant proteins and a liquid phase containing EBNAl protein or polypeptide, followed by subjecting the liquid phase containing EBNAl protein or polypeptide to a second ammonium sulfate treatment at an ammonium sulfate concentration which forms a solid phase containing EBNAl protein or polypeptide and a liquid phase containing contaminant proteins and then finally separating the solid phase containing EBNAl protein or polypeptide and the liquid phase containing contaminant proteins.
  • the first ammonium sulfate treatment is at a >0 to 30%, preferably 30%, ammonium sulfate concentration and the second ammonium sulfate treatment is at a 30 to 45%, preferably 45%, ammonium sulfate concentration.
  • the solid phase containing EBNAl protein or polypeptide is then purified, after separation, by affinity column chromatography, such as agarose-heparin column chromatography or oligonucleotide affinity column chromatography. By utilizing this purification process, it is believed that the recovered EBNAl protein is folded in its natural conformation.
  • insect cells preferably Sf-9 insect cells
  • EBNAl-containing recombinant baculovirus then harvested after a sufficient amount of time has passed to allow for protein expression.
  • the cytoplasmic membrane is disrupted and the nuclei containing expressed baculovirus-derived EBNAl protein or polypeptide ("bEBNAl") are pelleted to remove cytoplasm.
  • the nuclei are lysed, producing a viscous solution (“nuclear extract”) due to the presence of DNA.
  • the DNA is then removed by sonication which shears the DNA and partially reduces the viscosity of the nuclear extract.
  • a chromatography preparation solution is then added to the nuclear extract which is incubated and then centrifuged. This packs the DNA down tight into a small pellet, leaving most of the solution free of DNA.
  • the solution is decanted and then treated according to the above-described two-step ammonium sulfate precipitation procedure.
  • the centrifugation procedure after the second ammonium sulfate precipitation step produced a supernatant which is discarded and a pellet with bEBNAl.
  • the pellet containing bEBNAl is dissolved in a buffer and then dialyzed against the buffer. This dialyzed preparation is loaded onto an ion exchange chromatography column and eluted from it with a salt gradient and then purified using affinity column chromatography.
  • E. coli cells rather than insect cells, are used as host cells.
  • the present invention also relates to an isolated EBNAl protein or polypeptide formulation having substantially no components which generate false positive readings when used to detect EBV in human serum.
  • the isolated EBNAl protein or polypeptide of the present invention includes no more than 90%, preferably no more than 94%, of the Gly-Ala repeat amino acid sequence.
  • the present invention provides an isolated DNA molecule encoding EBNAl protein or polypeptide, a recombinant DNA expression system comprising an expression vector into which is inserted a heterologous DNA molecule encoding EBNAl protein or polypeptide, and a host cell, such as an insect cell, incorporating a heterologous DNA molecule encoding EBNAl protein or polypeptide, all of which have substantially no components which generate false positive readings when used to detect EBV in human serum.
  • the heterologous DNA molecule encoding the bEBNAl protein or polypeptide of the present invention comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows: ATG ACA GGA CCT GGA AAT GGC CTA GGA GAG
  • amino acid sequence corresponding to the DNA molecule of SEQ. ID. No. 1, is SEQ. ID. No. 2 as follows: Met Thr Gly Pro Gly Asn Gly Leu Gly Glu Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gin Arg Arg Gly Gly
  • Production of this isolated protein or polypeptide is preferably carried out using recombinant DNA technology.
  • the isolated DNA molecule is isolated from any other DNA molecule which expresses protein that generates false positive readings when the EBNAl protein or polypeptide is used to detect EBV in human serum.
  • the heterologous DNA molecule encoding the E. coli expression system-derived EBNAl protein or polypeptide ("eEBNAl") of the present invention comprises the nucleotide sequence corresponding to SEQ. ID. No. 3 as follows : ATG GGA GAA GGC CCA AGC ACT GGA CCC CGG
  • amino acid sequence corresponding to the DNA molecule of SEQ. ID. No. 3, is SEQ. ID. No. 4 as follows: Met Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly Gin Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp Phe Gly Lys His Arg Gly Gin
  • the heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • Recombinant genes may also be introduced into viruses, such as vaccina virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtll, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUCIS, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference) , pQE, pIH821, pGEX, pET series (see F.W.
  • viral vectors such as lambda vector system gtll, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC
  • Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al . , Molecular Cloning; A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982) , which is hereby incorporated by reference.
  • host-vector systems may be utilized to express the protein-encoding sequence (s) .
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus) .
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
  • SD Shine-Dalgarno
  • This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein.
  • the SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribcsomes by duplexing with the rRNA to allow correct positioning of the ribosome.
  • Promotors vary in their "strength" (i.e. their ability to promote transcription) .
  • strong promotors for the purposes of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene.
  • any one of a number of suitable promotors may be used. For instance, when cloning in E.
  • promotors such as the T7 phage promoter, lac promotor, trp promotor, recA pror.otor, ribosomal RNA promotor, the P R and P L promotors of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 ( tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside) .
  • IPTG isopropylthio-beta-D-galactoside
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
  • SD Shine-Dalgarno
  • Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant D ⁇ A or other techniques involving incorporation of synthetic nucleotides may be used.
  • Suitable host cells include, but are not limited to, bacteria, insect, virus, yeast, mammalian cells, and the like.
  • the present invention also provides a method of expressing an EB ⁇ A1 protein coding sequence in a cell.
  • an EB ⁇ A1 protein coding sequence is cloned into a baculovirus transfer vector.
  • the baculovirus transfer vector and Autographica calif ornica nuclear polyhedrosis genomic DNA are then co-transfected into insect cells, and recombinant baculoviruses are recovered. Cells are then infected with the recombinant baculovirus under conditions facilitating expression of isolated EBNAl protein or polypeptide in the cell.
  • the EBNAl protein coding sequence includes no more than 90%, preferably no more than 94%, of the Gly-Ala repeat amino acid sequence present in the naturally-occurring EBNAl protein coding sequence which spans the Gly-Ala repeat amino acid sequence.
  • the isolated EBNAl protein or polypeptide formulation of the present invention can be utilized for detection of EBV in a sample of human tissue or body fluids.
  • This detection process involves providing the isolated EBNAl protein or polypeptide formulation as an antigen, contacting the sample with the antigen, and detecting any reaction which indicates EBV is present in the sample using an assay system. More specifically, this technique permits detection of EBV in a sample of the following tissue or body fluids: blood, spinal fluid, sputum, pleural fluids, urine, bronchial alveolor lavage, lymph nodes, bone marrow, or other biopsied materials.
  • the assay system has a sandwich or competitive format.
  • Suitable assays include an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion precipitin reaction assay, an immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, a protein A immunoassay, or an immunoelectrophoresis assay.
  • the wild-type baculovirus, Autographica calif ornica nuclear polyhedrosis virus (AcMNPV) , and Spodcptera frugiperdi (Sf-9) cells used to propogate the baculoviruses, were kindly provided by Dr. Ora Rosen (Sloan- Kettering Cancer Center), with permission from Dr. Max D. Summers (Texas A & M University) .
  • Sf-9 cells were grown as monolayer cultures in Grace's medium (Gibco Laboratories) with 0.33% yeastolate and 0.33% lactalbumin hydrolysate (Difco) supplemented with 10% fetal bovine serum.
  • pVL941-SW (see Figure l) was constructed from pVL941 by Dr. Susan Wente in Dr. Ora Rosen's laboratory, by insertion of an Ncol / Xbal / Spel linker into the BamHI site of the polyhedrin gene in pVL941.
  • Plasmid pGEMoriP7 was constructed by ligating i?saI/HindIII DNA linkers to the ends of the Rsal fragment of p220.2 (kindly provided by Dr.
  • pGEMoriP was constructed from pGEMoriP7 using AccI to excise 2 kilobase pairs of DNA containing the EBNAl gene followed by religation to give pGEMoriP, which contains the entire oriF sequence.
  • AcMNPV-EBNAl Baculovirus
  • the EBNAl gene was excised from p205 using Rsal and Ball, which remove the first seven codons.
  • the initiating methionine was regenerated upon ligation into the baculovirus transfer vector pVL941-S to yield pVL941/EBNAl (Fig. 1) .
  • pVL941/EBNAl and AcMNPV DNA were cotransfected into Sf-9 insect cells by the calcium phosphate precipitation method as described by Summers et al . , Tex. Agric. Exp. Stn. Bull., 1555:27-31 (1987) , which is hereby incorporated by reference.
  • Virus from one of the resulting recombinant plaques was amplified in Sf-9 cells. Total DNA was prepared from these cells, digested with restriction enzymes, and analyzed by Southern blot hybridizations to verify the presence of the complete EBNAl Rsal-Ball fragment in the recombinant virus.
  • the oligonucleotide affinity column used in the procedure of Frappier, et al . , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus," The Journal of Biological Chemistry, 266 (12) :7819-26 (1991) was very difficult to synthesize and to use. It had very low binding capacity and each prep, needed to be run over the column in several batches. The bEBNAl that eluted was thus quite dilute and needed to be concentrated using either a heparin column or a MonoQ column. The following is an account of how to synthesize this improved column.
  • oligonucleotide sequences were: OLIGOl 5'Biotin-GGGAAGCATATGCTACCC-3' (SEQ. I.D. No. 5) ; and OLIG02 5' -GGGTAGCATATGCATATGCTTCCC-3' (SEQ. I.D. No. 6) .
  • 350 nmole of oligol and 440 nmole of oligo2 were mixed in 20ml of 10 mM Tris-HCl (pH 7.2) , 0.3 M NaCl, and 0.03 M sodium citrate (final pH 8.5) . The reaction was heated to 95°C for two minutes and allowed to cool to room temperature.
  • oligonucleotide was incubated with 20 ml of a 1:1 slurry of strepavadin beads (Sigma Chemical Company) and rotated end over end for 12 hours at 4°C. The solution was then placed into a glass column, the beds were allowed to settle, followed by an extensive wash to remove unreacted oligonucleotide with 20 mM Hepes (pH 7.5), 0.5 mM EDTA, 10% glycerol, and 350 mM NaCl. This column had a capacity of approximately 0.7 mg of bEBNA-1 per ml of packed beads.
  • bEBNAl was followed and quantitated by its ability to specifically retain a 900-bp fragment of oriP containing 20 copies of the 30-bp repeated sequence (family of repeats fragment) onto nitrocellulose filters.
  • the family of repeats fragment was excised from pGEMoriP with £coRI and Ncol , purified by agarose gel electrophoresis followed by electroelution, and quantitated by measuring the absorbance at 260 nm.
  • the oriP repeat fragment was end-labeled by filling in the 3 ' -recessed ends using the Klenow fragment of DNA polymerase I with four dNTPs and [ ⁇ - 32 P]TTP. Assays for bENBAl were performed by - 28
  • a 300-bp fragment of oriP containing the dyad and its four associated EBNAl binding sites was incubated with bENBAl as described for the family of repeats.
  • This dyad symmetry element fragment was excised from pGEMoriP with Hindlll and EcoRV, gel-purified, quantitated by absorbance at 260 nm, and end-labeled as described for the family of repeats fragment.
  • a 2-kilobase pair DNA fragment containing oriP was prepared from pGEMoriP and end-labeled near the dyad. This fragment was prepared by linearizing pGEMoriP with Hindlll, filling in the 3 ' -recessed ends with [ ⁇ - 32 P]TTP using the Klenow fragment of DNA polymerase I, then digesting with BamHI.
  • the HindiII to BamHI fragment containing the complete oriP sequence was gel-purified and incubated with bEBNAl as described for the family of repeats fragment .
  • Sf-9 cells (2.8 x 10 8 cells, 10 x 150-cm 2 flasks) were infected with recombinant EBNAl baculovirus as described herein. Twenty-four hours post-infection, the media was replaced with phosphate-free or methionine-free Grace's media (Gibco) supplemented with 0.33% lactalbumin hydrolysate and 1 mCi of [ 32 P] orthophosphate or [ 35 S] methionine (Du Pont-New England Nuclear) . Cells were labeled for 18 h before nuclei were prepared. Labeled bEBNAl was purified as described herein.
  • the sedimentation coefficient of bENBAl was measured by layering 40 ⁇ g of bEBNAl either alone or along with 60 ⁇ g of molecular weight standards (apoferritin, IgG, bovine serum albumin, avalbumin, and myoglobin) in 200 ⁇ l of 25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.5 mM EDTA, 10% glycerol onto 12-ml 10-30% glycerol gradients containing 25 mM Tris-HCl (pH 7.5) , 300 mM NaCl, 0.5 mM EDTA. Gradients were spun for 40 h at 270,000 x g at 5°C in a TH-641 rotor. After centrifugation, fractions of 160 ⁇ l were collected from the bottom of each tube.
  • molecular weight standards apoferritin, IgG, bovine serum albumin, avalbumin, and myoglobin
  • the Stokes radius of bENBAl was determined by injecting 40 ⁇ g of bEBNAl along with 60 ⁇ g of molecular weight standards in 200 ⁇ l of 25 mM Tris-HCl (pH 7.5) , 300 mM NaCl, 0.5 mM EDTA, 10% glycerol onto a 30-ml fast protein liquid chromatography Superose 12 gel filtration column. The column was developed in the same buffer. Fractions of 160 ⁇ l were collected. Two microliters of each fraction from the glycerol gradients and gel filtration columns were assayed for the presence of bEBNAl using the nitrocellulose filter binding assay described above. bENBAl and the molecular weight standards were visualized after SDS- polyacrylamide gel electrophoresis analysis by staining with Coomassie Blue. 31 -
  • the molar quantity of DNA in each fraction was measured upon diluting 100 ⁇ l of column fraction with 400 ⁇ l of column buffer and measuring the absorbance at 260 nm (assuming 1 absorbance unit equals 50 ⁇ l/ml DNA) . Approximately 90% of the radioactivity and absorbance at 260 nm was recovered after gel filtration.
  • the 300-bp Hindlll to Ec ⁇ RV fragment of pGEMoriP containing the dyad symmetry element was end-labeled using the Klenow fragment of DNA polymerase I and [ ⁇ - 32 P]TTP to fill in the Hindlll end of the fragment.
  • bEBNAl was incubated with 10 fmol of the 32 P-labeled dyad fragment in a 20- ⁇ l reaction containing 50 mM HEPES (pH 7.5) , 300 mM NaCl, 5 mM MgCl 2 for 10 min at room temperature. The reactions were then diluted to 50 mM NaCl and incubated with 30 units of Aval at 37 °C for 3 min.
  • the EBNAl gene was excised from plasmid p205 and inserted into the pVL941-SW baculovirus transfer vector as described more fully above and as shown in Fig. 1.
  • the resulting plasmid, pVL941-EBNAl contained the EBNAl gene, which translates into a 50 kDa protein lacking six amino- terminal amino acids and approximately 232 contiguous Gly- Ala residues of the Gly-Ala repeat region. Of these 232 amino acid residues, 6 were downstream of the Gly-Ala repeat such that there are still 13 of the 239 Gly-Ala residues remaining, representing 5.44%. Neither of these regions was essential for EBNAl-dependent replication in vivo when tested separately.
  • a recombinant baculovirus (AcMNPV-EBNAl) containing the EBNAl gene controlled by the strong polyhedrin gene promoter.
  • the EBNAl protein or polypeptide produced by Ac-MNPV-EBNAl is bENBAl.
  • bEBNAl is not a fusion protein, as the ENBA1 gene was placed directly adjacent to the only ATG sequence present in the 5' region of the polyhedrin gene in pVL94l-SW (Fig. 1) .
  • Sf-9 cells were seeded into 16 150-cm 2 culture flasks (3 x 10 7 cells/flask) (Corning) , allowed to attach, then infected with AcMNPV-EBNAl at a multiplicity of infection of three.
  • the cells were harvested 46 h post- infection, washed in 250 ml of ice-cold phosphate-buffered saline, and resuspended on ice in 70 ml of hypotonic buffer (20 mM HEPES (pH 7.5) , 1 mM MgCl 2 , 1 mM PMSf) using a Dounce homogenizer with pestle B.
  • Nuclei were collected upon centrifugation at 1000 x g for 10 min at 5°C, washed in 70 ml cf cold hypotonic buffer, and resuspended with the Dounce homogenizer and pestle B in 20 ml of 20 mM HEPES (pH 7.5) , 1 M NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM MgCl 2 , 1 mM PMSF, followed by incubation for 1 h on ice. This nuclear extract is sonicated for 2 minutes to shear the DNA and partially reduce the viscosity.
  • a solution of 5% Poly in P * Polyethylenimine, average molecular weight 50,000, Sigma Chemical Co., St.
  • the preparation After slowly stirring for 1 hour at 4 °C, the preparation is spun at 15,000 rpm for 30 minutes at 4 °C. The supernatant is decanted and then ammonium sulfate is added to a final saturation of 45% (e.g., adding 7.8 ml of 100% saturated ammonium sulfate solution) in order to bring down the bEBNAl, yet leave other contaminants in solution. After slowly stirring for 1 hour at 4 °C, the preparation is spun for 30 minutes at 15,000 rpm at 4 °C.
  • the bEBNAl protein-containing pellet is then dissolved in buffer A (20 mM Hepes (pH 7.5) , 0.5 mM EDTA, 2 mM DTT, 1 mM PMSF, 20% glycerol) and dialyzed against 2 liters of buffer A for 4 hours at 4 °C and then against another 2 liters of buffer A overnight before loading onto a 30-ml heparin-agarose column (Bio-Rad) .
  • buffer A (20 mM Hepes (pH 7.5) , 0.5 mM EDTA, 2 mM DTT, 1 mM PMSF, 20% glycerol
  • the 2 M NaCl eluate containing 33% of the oriP binding activity (50 ml) was dialyzed against 500 mM NaCl, diluted with buffer A to a conductivity equivalent to 260 mM NaCl (105 ml) , and loaded onto a 1-ml Mono Q column.
  • bEBNAl was eluted with buffer A containing 500 mM NaCl. Aliquots of active fractions (20 ⁇ l/tube) were stored at -70°C.
  • the bEBNAl can be concentrated by diluting the preparation with buffer A to a conductivity in the range of 250-300 mM NaCl and loaded onto a 1 ml Heparin Agarose column followed by elution using buffer A containing IM NaCl.
  • Fraction Protein Activity Specific Purification Yield Activity mg units units/mg -fold %
  • This modified protocol gives about a 5-fold higher amount of the bEBNA-1 at the end of the procedure.
  • the greater amount is probably due to recovery of more bEBNAl from the nucleus due to the elimination of DNA using Polyamine P instead of high speed centrifugation. In effect, one obtains much more solution phase due to tight compaction of the DNA by Polyamine P.
  • the purity at the end is undoubtedly better than in the previous protocol due to the ammonium sulfate cut, but it cannot be detected by specific activity, because the difference is only between 95% and 98% (or greater) purity.
  • this product in an ELISA assay one never knows when a very small level of impurity will invalidate the assay. Thus, the more pure - the better - even if it is a difference in going from 98 to 99 percent.
  • Homogeneous bEBNAl was assayed for the ability to hydrolyze ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and TTP in 1, 3, and 10 mM MgCl 2 , in the absence of DNA and in the presence of either oriP-containing duplex DNA or single- stranded DNA.
  • Nucleotide hydrolysis assays were performed by incubating 200 ng of bEBNAl with 50 ⁇ M [o;- 32 P] - or [ ⁇ - 32 P] nucleoside triphosphate and deoxynucleoside triphosphate in 10 ⁇ l of 20 mM Tris-HCl (pH 7.5) and 1, 3, or 10 mM MgCl 2 for 30 min at 37°C.
  • the T subunit of Escherichia coli DNA polymerase III holoenzyme was used as a positive control for ATP hydrolysis according LO the method of Tsuchihashi et al . , J. Biol . Chem. , 264:17790-17795 (1989) , which is hereby incorporated by reference. No hydrolysis of any nucleoside triphosphate by bEBNAl was detected (data not shown) .
  • bEBNAl was tested in the standard oligonucleotide displacement type of helicase assay according to Matson, J. Biol . Chem. , 261:10169-10175 (1986), which is hereby incorporated by reference. bEBNAl was examined for an ability to displace, from single-stranded circular bacteriophage ⁇ X174 DNA, a 32 P- end-labeled flush DNA 30-mer, a 5' -tailed DNA 30-mer, and a 3 '-tailed DNA 46-tner.
  • Each helicase substrate was then purified from unhybridized oligonucleotide by gel filtration on Bio-Gel A- 1.5m Helicase assays were performed by incubating 400 ng of bEBNAl with 9 fmol of DNA substrate in 30 mM HEPES (pH 7.5) , 4 mM ATP, 7 mM MgCl 2 , 1 mM dithiothrietol for 30 min at 37°C. Positive control reactions contained 400 ng of SV40 large T antigen. Reaction products were analyzed for oligonucleotide displacement on a 15% polyacrylamide gel. The SV40 large T antigen was used as a positive control according to the method of Goetz et al. , J.
  • bEBNAl was labeled in vivo with [ 32 P] orthophosphate and purified to homogeneity.
  • bEBNAl was the major 32 P- labeled protein in the nuclear extract and was not detected in the cytoplasm (Fig. 3) .
  • Treatment of pure [ 32 P] bEBNAl with CIP resulted in loss of all detectable radioactive phosphate from bEBNAl (Fig. 3) . Since CIP has previously been shown to dephosphorylate serine residues only, Shaw et al . , Virology, 115:88-96 (1981) and Klausing et al, Virol .
  • bEBNAl is presumably phosphorylated only on serine. Further identification of phosphorylated residues in bEBNAl was performed by acid hydrolysis of [ 32 P]bEBNAl and separation of the phosphoamino acids by high voltage paper electrophoresis (Fig. 4) . Samples of [ 32 P]bEBNAl hydrolyzed for 1, 2, and 4 h were analyzed to ensure identification of any [ 32 P]phosphothreonine, which requires longer hydrolysis times, or [ 32 P]phosphotyrosine, which is less stable to acid hydrolysis according to the method of Cooper et al . , Methods Enzvmol ..
  • bEBNAl was analyzed by glycerol gradient sedimentation; an s value of 4.6 was obtained by comparison with protein markers with known s values (Fig. 3A) .
  • a Stokes radius of 50 A for bENBAl was determined by gel filtration analysis and comparison with protein standards of known Stokes radius (Fig. 3B) .
  • oriP binding activity co-eluted with the bEBNAl protein visualized in SDS-polyacrylamide gel analysis of the column fractions (data not shown) .
  • the s value and Stokes radius were combined in the equation of Siegel et al . , Biochim. Biophys.
  • 35 S-Labeled bEBNAl protein was prepared in vivo by metabolic labeling using [ 35 S] methionine followed by purification to homogeneity.
  • the [ 35 S]bEBNAl was used to measure the number of bEBNAl molecules bound to oriP under conditions of saturating bEBNAl.
  • a plasmid containing the complete oriP sequence was incubated with increasing amounts of [ 35 S] bEBNAl then gel-filtered to separate [ 35 S] bEBNAl bound to DNA in the excluded fractions from the unbound [ 35 S]bEBNAl in the included fractions.
  • bEBNAl 50 ng was incubated with 40 fmol of 32 P- labeled dyad fragment or 32 P-labeled repeat fragment in various concentrations of NaCl and in the presence of excess (2.5 ⁇ g) calf thymus DNA (Fig. 7) .
  • the binding profile indicates that the specific interaction of bEBNAl with the dyad symmetry element was maximum at 250-300 mM NaCl and dropped off sharply at higher NaCl concentrations.
  • Binding of bEBNAl to the family of repeats remained stable up to 500 mM NaCl.
  • the relative binding strength of bEBNAl for the family of repeats versus the dyad symmetry element depended on the salt concentration.
  • the apparent requirement of high salt for binding bEBNAl to labeled DNA in these experiments may be attributed to efficient competition by nonspecific calf thymus DNA at low NaCl concentration.
  • bEBNAl The interaction of bEBNAl with the family of repeats and dyad symmetry element of oriP was also assessed by examining the amount of bEBNAl required to retain each element on nitrocellulose filters. Increasing amounts of bEBNAl were incubated with 10 fmol of 32 P-end-labeled repeat or dyad DNA fragment in 20 ⁇ l of buffer containing 300 mM
  • the apparent K d for bEBNAl binding to the dyad symmetry element calculated from these data is 2 nM (assuming four bEBNAl dimers were bound per dyad symmetry element) .
  • the family of repeats was retained onto nitrocellulose at lower levels of bEBNAl than required for binding the dyad symmetry element (Fig. 8, open circles) .
  • An apparent K d for bEBNAl binding to the family of repeats was calculated to be 0.2 nM (assuming four bEBNAl dimers were bound per family of repeats) .
  • the binding of bEBNAl to the dyad symmetry element was further examined by an Aval endonuclease protection assay.
  • An Aval site was present at the junction of two of the four EBNAl binding sites in the dyad symmetry element (Fig. 9) .
  • Increasing amounts of bEBNAl were incubated with 10 fmol of the dyad symmetry element, end-labeled with 32 P at one end only. The reaction was then treated with sufficient Aval to completely digest the DNA within 3 min at 37°C. Digestions were stopped with SDS and subjected to polyacrylamide gel electrophoresis to separate DNA fragments cut by Aval from uncut (Aval-protected) DNA (Fig. 9) .
  • the Aval protection analysis showed that a 20-fold molar excess of bEBNAl dimers (200 ng in Fig. 9) was required over the dyad fragment to detect protection of the Aval site, followed by a very sharp increase in protection against Aval at levels above 20 bEBNAl dimers per dyad symmetry element .
  • the small difference between the Aval protection assay (Fig. 9) and the nitrocellulose filter binding assay (Fig. 8) showed approximately 1.5 times more bEBNAl was needed to bind the dyad symmetry element onto a nitrocellulose filter relative to the amount of bEBNAl needed to protect the Aval site.
  • dyad symmetry element is accompanied by the family of repeats within oriP which may affect the interaction of EBNAl with the dyad symmetry element in the complete oriP sequence.
  • bEBNAl was incubated with oriP labeled with 32 P at the end near the dyad.
  • the family of repeats was separated from the dyad symmetry element by digestion with EcoRV (Fig. 8) for each assay an aliquot was removed prior to filtration, quenched with SDS (i.e., sodium dodecyl sulfate) , and analyzed in an agarose gel to confirm that EcoKV had completely separated the dyad from the oriP DNA.
  • SDS i.e., sodium dodecyl sulfate
  • a less stable complex of bEBNAl with the dyad may assemble in the presence of the family of repeats.
  • the nonessential region of oriP between the family of repeats and dyad symmetry element may influence the nitrocellulose binding assay, or the presence of the dyad may cause more cooperative binding of bEBNAl to the family of repeats, effectively decreasing the availability of bEBNAl for binding the dyad.
  • EBNAl the viral encoded protein which binds the latent phase origin ( oriP) of EBV, in the baculovirus system and its purification of homogeneity.
  • oriP latent phase origin
  • replication initiation in the dyad is greatly stimulated by the family of repeats. .Id.
  • One mechanism by which the repeats might activate the dyad is by altering the interaction of EBNAl with the dyad symmetry element .
  • the nitrocellulose filter binding assay suggested that the family of repeats reduced the concentration of bEBNAl required to initiate binding to the dyad of bEBNAl required to initiate binding to the dyad symmetry element. If the interaction of EBNAl with the dyad symmetry element is important for the initiation of replication from oriP, then the stimulation of dyad binding by the family of repeats at low EBNAl concentration may be one mechanism by which the repeats enhance replication from oriP.
  • EBNAl is essential for latent EBV replication, yet the precise biochemcal function of EBNAl remains elusive.
  • the bEBNAl protein should prove useful in biochemical assays to analyze the mechanism by which EBNAl activates oriP to function as an origin of replication, a plasmid maintenance element, and a transcriptional enhancer. See Yates et al . , Cancer Cells, 6:197-205 (1988) , which is hereby incorporated by reference. Applicant finds no ATPase (or other nucleoside triphosphatase) , helicase, ligase, topoisomerase, DNA polymerase, oxonuclease, or endonuclease activities associated with bEBNAl.
  • EBNAl plays a different role in replication than the large T antigen of SV40. It is always possible', however, that the true activity of EBNAl will only be revealed upon binding other proteins or by modification at a specific site(s) . Furthermore, the possibility cannot be excluded that, although the six amino-terminal amino acids and glycine-alanine repeat region of EBNAl, lacking in bEBNAl, are nonessential for EBNAl function in vivo, id. , they may affect the biochemical activity of EBNAl in vi tro. Elucidation of the precise role of EBNAl in replication and the mechanism(s) of replication control at oriP would be greatly facilitated by development of an in vi tro system capable of initiating replication from oriP.
  • the DNA template used for the sequence analysis of the GlyAla deletion was the 10.6 kb bEBNAl baculovirus transfer vector, called pVL941-EBNAl, the construction of which was described in L. Frappier, et al . , "Overproduction, Purification, and Characterization of EBNAl, the Origin Binding Protein of Epstein-Barr Virus, " J. Biol. Chem. 766 (12) : 7819-26 (1991) .
  • the sequencing primer used in this analysis was positioned 187 nucleotides in from the A of the ATG start codon of EBNAl; the sequence of the sequencing primer was 5 'AAAAACGTCCAAGTTGCATTG-3 ' (SEQ. ID. No. 7) . Sequencing was performed using the Sequenase based protocol and version 2 kit of United States Biochemical, Cleveland, Ohio according to the manufacturers specifications.
  • the gene and expression plasmid were constructed by PCR using the following primers: N - terminus - 5' - GAT CGG CAT ATG GGA GAA GGC CCA AGC ACT GGA - 3' (the underline is the Met for the first amino acid, and the GGA that follows encodes amino acid 442 of EBNAl) (SEQ. ID. No. 8) ; and C - terminus - 5' - CT GGT GGA TCC TTA ACC AAC AGA AGC ACG ACG CAG CTC CTG CCC TTC CTC AC - 3 ' (the underlined coder, encodes the last amino acid of the eEBNAl) (SEQ. ID. No. 9) .
  • the template used in the PCR reaction was p291 (FIG. 11) , a plasmid containing the entire EBNAl gene (see FIGS. 12A-C) .
  • the cycling conditions were 94 °C, 30 sec./ 60 °C, 30 sec./ 72 °C, 60 sec.
  • This cycle is repeated 30 times in 100 ⁇ liters of 10 mM Tris-HCl (pH 8.3) , 50 mM KCl, 1.5 mM MgCl ; , 200 ⁇ molar each dATP, dCTP, dGTP, dTTP, 0.01% gelatin, 2.5 units TagI polymerase (Perkin-Elmer Cetus) , 1 ⁇ molar of each primer (described above) , and 1 ng of plasmid p291.
  • the 641 bp fragment was purified by phenol extraction in 2% SDS followed by sequential digestion with 10 units of Ndel (New England Biolabs) and then 10 uni ⁇ s of BamHI (New England Biolabs) .
  • Ndel/Ba HI 624 bp fragment (see SEQ. ID. NO. 3) was purified from an agarose gel and ligated into pET3c (digested with Ndel and BamHI) to yield pET-eEBNAl, as shown in Figure 10. Sequence analysis confirmed that no errors had been introduced by PCR amplification.
  • the pET-eEBNAl plasmid was transformed into E. coli strain BL21 (DE3)pLysS and the cells were grown at 37 °C in 4 liters of LB medium (per liter: lOg Bacro-tryptone, 5g Bacto-yeast, lOg NaCl, pH 7.5) supplemented with 1% glucose, 10 ⁇ g/ml thiamine, 50 ⁇ g/ml thyrr.ine, 100 ⁇ g/ml ampicillin, and 30 ⁇ g/ml chloramphenicol . Upor. reaching an absorbance at 600 nm of 0.8, IPTG was added to 0.4 mM, and after 2 hours at 37 °C, the cells were harvested by centrifugation (I5g net weight) .
  • LB medium per liter: lOg Bacro-tryptone, 5g Bacto-yeast, lOg NaCl, pH 7.5
  • glucose 10 ⁇ g/ml thiamine
  • the cells were frozen at -70 °C and then thawed to 4 °C, and then resuspended in 40 ml of 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 50 mM glucose. At this point, the cells lyse due to the lysozyme produced by the pLysS plasmid and the freeze-thaw procedure.
  • the volume was brought to 100 ml using solution I and the DNA removed by precipitation by adding 10 ml NaCl, 1.4 ml of 5% Polymin P * (50 kDa) dissolved in 20 mM Tris-HCl (pH 7.5) . After stirring slowly for 30 minutes at 4 °C, the precipitation was spun at 18,000 rpm at 4 °C.
  • the supernatant (82 ml) was adjusted to 70% ammonium sulfate by adding 191 ml of 100% saturated ammonium sulfate to precipitate the eEBNAl protein.
  • the eEBNAl- containing precipitate was then pelleted by centrifugation for 30 minutes at 1,000 rpm in the GSA rotor at 4 °C.
  • the pellet was dissolved in 40 ml of buffer B (20 mM Tris-HCl (pH 7.5) , 0.5 mM EDTA, 2 mM DTT, 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride (i.e., PMSF) ) and loaded onto a 330 ml column of Bio-Gel P-6 equilibrated in buffer B. Fractions of 8 ml were collected at a flow rate of 3 ml/minute and assayed for total protein by the Bradford reagent (Bio-Rad) . Peak fractions (11-29) are pooled (700 mg protein) .
  • buffer B 20 mM Tris-HCl (pH 7.5) , 0.5 mM EDTA, 2 mM DTT, 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride (i.e., PMSF)
  • the 700 mg protein pool was loaded onto a 320 ml column of Heparin-Agarose (Bio-Rad) equilibrated in buffer B.
  • the column was eluted with a 3.2 liter linear gradient of buffer B from 0 mM NaCl to 800 mM NaCl. Fractions of 26 ml were collected and assayed for total protein and for eEBNAl.
  • the eEBNAl eluted in fractions 60-96 and these were pooled (39 mg) and precipitated by adding 434g solid ammonium sulfate (70% saturation) .
  • the protein precipitate was collected by centrifugation, resuspended in 20 ml buffer B, and dialyzed against 2 liters of buffer B for 4 hours and then against another 2 liters of buffer B overnight.
  • the dialysate was loaded onto a 40 ml column of Q Sepharose (Pharmacia) equilibrated in buffer B.
  • the eEBNAl was eluted with a linear gradient of 400 ml of 0 mM NaCl to 800 ml NaCl in buffer B. Fractions of 5 ml were collected at a flow rate of 1 ml/minute and the fractions were assayed for eEBNAl.
  • Fractions containing eEBNAl were pooled (fractions 34-44, 20 mg total) .
  • This eEBNAl-containing pool had a conductivity equal to 386 mM NaCl and was diluted with buffer B to a conductivity equal to 48 mM NaCl, then loaded onto a 4 ml column of CM Sepharose (Pharmacia) equilibrated in buffer B.
  • the eEBNAl was eluted using a 40 ml linear gradient of 0 mM NaCl to 700 mM NaCl in buffer B and the fractions containing eEBNAl were pooled (fractions 24-34, 18 mg total) and dialyzed against buffer B and stored frozen at -70 °C.
  • ADDRESSEE Nixon, Hargrave, Devans & Doyle
  • B STREET: Clinton Square, P.O. Box 1051
  • GCAGATCACC CAGGAGAAGG CCCAAGCACT GGACCCCGGG GTCAGGGTGA TGGAGGCAGG 660 CGCAAAAAAG GAGGGTGGTT TGGAAAGCAT CGTGGTCAAG GAGGTTCCAA CCCGAAATTT 720
  • Pro Gly Ala lie Glu Gin Gly Pro Ala Asp His Pro Gly Glu Gly Pro 195 200 205
  • Glu Asn lie Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val 245 250 255 Glu Arg Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO: 8 :

Abstract

L'invention se rapporte à un procédé d'expression et de récupération d'une protéine ou d'un polypeptide (EBNA1) de l'antigène nucléaire 1 du virus Epstein Barr consistant à traiter des cellules dont le noyau contient la protéine ou le polypeptide EBNA1 exprimé afin de récupérer le noyau contenant la protéine ou le polypeptide EBNA1 exprimé. Le noyau contenant la protéine ou le polypeptide EBNA1 exprimé est ensuite séparé dans une fraction liquide contenant la protéine ou le polypeptide EBNA1 exprimé et dans une fraction liquide contenant pratiquement tout l'ADN provenant du noyau. La fraction liquide est séparée de la fraction solide, et la protéine ou le polypeptide EBNA1 est récupéré à partir de la fraction liquide. L'invention se rapporte également à une protéine ou un polypeptide EBNA1 ne possédant pas de composants générant des lectures faux positif lorsqu'ils sont utilisés pour détecter le virus Epstein Barr dans le sérum humain, à la molécule d'ADN codant cette protéine et à l'expression par recombinaison de cette protéine. Cette protéine est utilisée dans un procédé de détection du virus Epstein Barr.
PCT/US1995/008700 1994-07-13 1995-07-13 Proteine de l'antigene nucleaire 1 du virus epstein barr, son expression et sa recuperation Ceased WO1996002563A1 (fr)

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KR101796731B1 (ko) 2016-10-11 2017-11-13 창원대학교 산학협력단 열차폐 코팅용 코어/쉘 복합체 분말의 제조방법 및 이에 의해 제조된 코어/쉘 복합체
KR102765079B1 (ko) * 2021-09-17 2025-02-06 울산과학기술원 고체 큐빗 이미징 장치

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