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 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%.
Meij
P
P
Department of Pathology, Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands. p,meij@azvu.nl
Vervoort
M B
MB
de Gooijer
K
K
Bloemena
E
E
Meijer
C J
CJ
Middeldorp
J M
JM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D019149:Bioreactors; D002460:Cell Line; D002470:Cell Survival; D002846:Chromatography, Affinity / Q000379:methods; D046508:Culture Techniques / Q000379:methods; D004247:DNA / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015151:Immunoblotting; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D019309:Epstein-Barr Virus Nuclear Antigens; D011994:Recombinant Proteins; D004247:DNA