Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1978 Sep;27(3):604–611. doi: 10.1128/jvi.27.3.604-611.1978

Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines.

J Luka, T Lindahl, G Klein
PMCID: PMC525848  PMID: 212583

Abstract

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.

Full text

PDF
604

Images in this article

609Image
on p.609
610Image
on p.610

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baron D., Strominger J. L. Partial purification and properties of the Epstein-Barr virus-associated nuclear antigen. J Biol Chem. 1978 Apr 25;253(8):2875–2881. [PubMed] [Google Scholar]
  2. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  3. Epstein M. A., Achong B. G., Barr Y. M., Zajac B., Henle G., Henle W. Morphological and virological investigations on cultured Burkitt tumor lymphoblasts (strain Raji). J Natl Cancer Inst. 1966 Oct;37(4):547–559. [PubMed] [Google Scholar]
  4. Fluck M. M., Staneloni R. J., Benjamin T. L. Hr-t and ts-a: two early gene functions of polyoma virus. Virology. 1977 Apr;77(2):610–624. doi: 10.1016/0042-6822(77)90486-x. [DOI] [PubMed] [Google Scholar]
  5. Ito Y., Spurr N., Dulbecco R. Characterization of polyoma virus T antigen. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1259–1263. doi: 10.1073/pnas.74.3.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Klein G., Dombos L. Relationship between the sensitivity of EBV-carrying lymphoblastoid lines to superinfection and the inducibility of the resident viral genome. Int J Cancer. 1973 Mar 15;11(2):327–337. doi: 10.1002/ijc.2910110210. [DOI] [PubMed] [Google Scholar]
  7. Klein G., Giovanella B., Westman A., Stehlin J. S., Mumford D. An EBV-genome-negative cell line established from an American Burkitt lymphoma; receptor characteristics. EBV infectibility and permanent conversion into EBV-positive sublines by in vitro infection. Intervirology. 1975;5(6):319–334. doi: 10.1159/000149930. [DOI] [PubMed] [Google Scholar]
  8. Klein G., Lindahl T., Jondal M., Leibold W., Menézes J., Nilsson K., Sundström C. Continuous lymphoid cell lines with characteristics of B cells (bone-marrow-derived), lacking the Epstein-Barr virus genome and derived from three human lymphomas. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3283–3286. doi: 10.1073/pnas.71.8.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Lenoir G., Berthelon M. C., Favre M. C., de-Thé G. Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen. J Virol. 1976 Feb;17(2):672–674. doi: 10.1128/jvi.17.2.672-674.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lewis J. B., Atkins J. F., Baum P. R., Solem R., Gesteland R. F., Anderson C. W. Location and identification of the genes for adenovirus type 2 early polypeptides. Cell. 1976 Jan;7(1):141–151. doi: 10.1016/0092-8674(76)90264-6. [DOI] [PubMed] [Google Scholar]
  12. Luka J., Siegert W., Klein G. Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein. J Virol. 1977 Apr;22(1):1–8. doi: 10.1128/jvi.22.1.1-8.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Matsuo T., Nishi S., Hirai H., Osato T. Studies on Epstein-Barr virus-related antigens. II. Biochemical properties of soluble antigen in Raji Burkitt lymphoma cells. Int J Cancer. 1977 Mar 15;19(3):364–370. doi: 10.1002/ijc.2910190313. [DOI] [PubMed] [Google Scholar]
  14. Miller G., Lipman M. Release of infectious Epstein-Barr virus by transformed marmoset leukocytes. Proc Natl Acad Sci U S A. 1973 Jan;70(1):190–194. doi: 10.1073/pnas.70.1.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ohno S., Luka J., Lindahl T., Klein G. Identification of a purified complement-fixing antigen as the Epstein-Barr-virus determined nuclear antigen (EBNA) by its binding to metaphase chromosomes. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1605–1609. doi: 10.1073/pnas.74.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Reedman B. M., Klein G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer. 1973 May;11(3):499–520. doi: 10.1002/ijc.2910110302. [DOI] [PubMed] [Google Scholar]
  17. Ryan L. D., Vestling C. S. Rapid purification of lactate dehydrogenase from rat liver and hepatoma: a new approach. Arch Biochem Biophys. 1974 Jan;160(1):279–284. doi: 10.1016/s0003-9861(74)80035-4. [DOI] [PubMed] [Google Scholar]
  18. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  19. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES