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%.

Location and limitation of cellulose production by Acetobacter xylinum established from oxygen profiles.

The static fermentation of coconut water sucrose by Acetobacter xylinum was carried out at initial pH's of 3.0, 4.0, 5.0 or 6.0. Cellulose was produced at the surface, and its production was most favourable at pH's 4.0 and 5.0. These pH values also allowed for optimal bacterial growth. Oxygen concentration profiles were measured with microelectrodes at different cultivation stages, and steep profiles were obtained with penetration depths between 50 and 100 microm. A substrate penetration depth analysis confirmed the hypothesis that the first stage of the fermentation is entirely oxygen controlled. Diffusion calculations showed, however, that at a later stage sucrose becomes a limiting substrate also, which was confirmed by the decrease in cellulose production rate over time. The effective diffusion coefficient of oxygen in deactivated cellulose pellicles was measured with microelectrodes, and a value of 1.4 x 10(-9) m2/s was obtained under all investigated conditions. The oxygen flux was 5.9 x 10(-6) mol/m2.s, while a significantly higher value of 9.1 x 10(-6) mol/m2.s was obtained at pH 4.0.

Membrane bioreactor with a porous hydrophobic membrane as a gas-liquid contactor for waste gas treatment.

A novel type of bioreactor for waste gas treatment has been designed. The reactor contains a microporous hydrophobic membrane to create a large interface between the waste gas and the aqueous phase. To test the new reactor, propene was chosen because of its high air/water partition coefficient, which causes a low water concentration and hampers its removal from air. Propene transfer from air to a suspension of propene-utilizing Xanthobacter Py2 cells in the membrane bioreactor proved to be controlled by mass transfer in the liquid phase. The resistance of the membrane was negligible. Simulated propene transfer rates agreed well with the experimental data. A stable biofilm of Xanthobacter Py2 developed on the membrane during prolonged operation. The propene flux into the biofilm was 1 x 10(-6) mol m(-2) s(-1) at a propene concentration of 9.3 x 10(-2) mol m(-3) in the gas phase. (c) 1995 John Wiley & Sons, Inc.