Bioprocess applications of a Sindbis virus‐based temperature‐inducible expression system
Boorsma M., Hoenke S., Marrero A., Fischer R., Bailey JE., Renner WA., Bachmann MF.
AbstractThe production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature‐regulatable Sindbis virus replicon‐based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth‐inhibiting, anti‐viral protein interferon β (β‐IFN). Selected clones were analyzed for temperature‐dependent β‐IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 × 105 IU/106 cells/day (0.5 μg/106 cells/day) in suspension cultures and over 1.5 × 106 IU/mL/day (7.5 μg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine‐tagged β‐IFN purified from T‐flask cultures was highly glycosylated and showed high specific activity. β‐IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS‐inducible expression system for the production of high‐quality glycoproteins in different reactors. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 602–609, 2002.