Bioreactor-scale production and one-step purification of Epstein-Barr nuclear antigen 1 expressed in baculovirus-infected insect cells

P Meij, M B Vervoort, K de Gooijer, E Bloemena, C J Meijer, J M Middeldorp

Research output: Contribution to journalArticleAcademicpeer-review

7 Citations (Scopus)


Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated malignancies and is essential for EBV-genome maintenance. Antibodies to EBNA1 are abundantly detected in serum of most EBV carriers but EBNA1 escapes recognition by effector T-lymphocytes. To further study the functional and immunological characteristics of EBNA1 it is important to have sufficient quantities of purified EBNA1 available. This paper describes a simple, reproducible method for the production and purification of EBV-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of EBNA1 expression levels in cell lines and for monitoring bEBNA1 purification and overall yields we developed a quantitative and EBNA1-specific capture ELISA. We observed that EBV-positive cell lines express EBNA1 at different levels, with the B cell lymphoblastoid cell line X50/7 having the highest production. However, much larger quantities (380-fold) were obtained by expressing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling-up experiments revealed that bEBNA1 expression kinetics and protein stability are identical in 1-liter stirred bioreactors when compared to expression in stationary culture flasks. Optimal expression was reached after 72 h following inoculation at 1 pfu/cell, when insect cell viability was about 50%. For purification the nuclear fraction containing most of the bEBNA1 (>95%) was isolated. Solubilized bEBNA1 was purified by a one-step oriP DNA-Sepharose affinity purification procedure, using biotinylated PCR-amplified family of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an estimated purity of >95%.

Original languageEnglish
Pages (from-to)324-33
Number of pages10
JournalProtein Expression and Purification
Issue number2
Publication statusPublished - Nov 2000


  • Animals
  • Baculoviridae
  • Bioreactors
  • Cell Line
  • Cell Survival
  • Chromatography, Affinity
  • Culture Techniques
  • DNA
  • Enzyme-Linked Immunosorbent Assay
  • Epstein-Barr Virus Nuclear Antigens
  • Immunoblotting
  • Journal Article
  • Protein Binding
  • Recombinant Proteins
  • Sensitivity and Specificity
  • Spodoptera

Cite this