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.
Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Gaurav
G
Batra
Chandrasekhar
C
Gurramkonda
Satish Kumar
SK
Nemani
Swatantra Kumar
SK
Jain
Sathyamangalam
S
Swaminathan
Navin
N
Khanna
D018533:Biomass; D019149:Bioreactors; D002853:Chromatography, Liquid; D003470:Culture Media; D003716:Dengue Virus / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics*; D011995:Recombination, Genetic; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics*
D003470:Culture Media; D014764:Viral Proteins