Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.
J Biosci Bioeng. 2010 Oct;110(4):408-14. doi: 10.1016/j.jbiosc.2010.05.001. Epub 2010 Jun 2.
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.
我们构建了一株能够分泌登革病毒 2 型包膜结构域 III（sEDIII-2）的甲基营养型酵母毕赤酵母重组克隆。我们探索了各种诱导条件，包括培养基组成、温度、pH 值和甲醇浓度，以优化摇瓶培养中 sEDIII-2 表达的条件。在 20°C 下，在 pH5.8 缓冲的培养基中以 2%（v/v）甲醇诱导，可获得最高的 sEDIII-2 分泌量。通过重复诱导相同的初始生物量，产量可进一步提高至 70%。使用分批补料培养策略，我们观察到摇瓶中的产量可以在生物反应器中放大约 8 倍。经纯化后，我们获得了约 94%的纯度和＞70%的回收率。本研究首次证明了使用毕赤酵母宿主分泌包膜结构域 III 的可行性，这将对登革热疫苗的亚单位方法的发展具有重要意义。
