Optimization of conditions for secretion of dengue virus type 2 envelope domain III using Pichia pastoris.

We have developed a recombinant clone of the methylotrophic yeast Pichia pastoris capable of secreting dengue virus type 2 envelope domain III (sEDIII-2). We explored various induction parameters including media composition, temperature, pH, and methanol concentration, to optimize conditions for sEDIII-2 expression in shake flask culture. Induction at 20°C in the presence of 2% (v/v) methanol in a medium buffered to pH 5.8 resulted in highest secretion of sEDIII-2. This yield could be further enhanced up to 70% by repeated induction of the same initial biomass. Using a fed-batch cultivation strategy, we observed that shake-flask yields can be scaled up ∼8-fold in a bioreactor. We obtained ∼94% purity with >70% recovery after purification. This study, which demonstrates for the first time the feasibility of secreting envelope domain III using the P. pastoris host, will be relevant to sub-unit approaches to dengue vaccine development.