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

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 t o have sufficient quantities of purified EBNA1 available. This paper descri bes a simple, reproducible method for the production and purification of EB V-encoded EBNA1 expressed in insect cells (bEBNA1). For quantification of E BNA1 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 p roduction. However, much larger quantities (380-fold) were obtained by expr essing bEBNA1 in recombinant-baculovirus-infected Sf9 insect cells. Scaling -up experiments revealed that bEBNA1 expression kinetics and protein stabil ity are identical in 1-liter stirred bioreactors when compared to expressio n in stationary culture flasks. Optimal expression was reached after 72 h f ollowing 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-Sep harose affinity purification procedure, using biotinylated PCR-amplified fa mily of repeats (FR)-domain products immobilized onto streptavidin agarose. A >200-fold specific enrichment was reached and yields of bEBNA1 with an e stimated purity of >95%. (C) 2000 Academic Press.