Sinha Jayanta, Inan Mehmet, Fanders Sarah, Taoka Shinichi, Gouthro Mark, Swanson Todd, Barent Rick, Barthuli Ardis, Loveless Bonnie M, Smith Leonard A, Smith Theresa, Henderson Ian, Ross John, Meagher Michael M
Biological Process Development Facility, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0466, USA.
J Biotechnol. 2007 Jan 10;127(3):462-74. doi: 10.1016/j.jbiotec.2006.07.022. Epub 2006 Jul 31.
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.
已开发出一种在毕赤酵母中生产候选疫苗抗原——肉毒杆菌神经毒素血清型E重组C末端重链片段（rBoNTE(H(c))）的方法。将插入到pHILD4大肠杆菌 - 毕赤酵母穿梭质粒中的rBoNTE(H(c))基因转化毕赤酵母菌株GS115。对该克隆进行了遗传稳定性、拷贝数和BoNTE(H(c))序列的鉴定。在5升和19升规模的补料分批发酵工艺开发之前，在摇瓶中确认了来自Mut(+) HIS4克隆的rBoNTE(H(c))的表达。发酵过程包括在分批和补料分批模式下使用限定培养基的甘油生长阶段，随后是甘油/甲醇过渡阶段以适应在甲醇上生长，以及甲醇诱导阶段，从而产生rBoNTE(H(c))。比生长速率、生长与诱导阶段的比例以及诱导时间对于rBoNTE(H(c))的最佳生产和最小蛋白水解降解至关重要。使用计算机控制的指数生长模型进行工艺自动化，并使用尾气分析进行工艺监测。通过HPLC和蛋白质印迹分析确定，优化后的工艺在甲醇上的诱导时间为9小时，每克湿细胞质量最多可产生3毫克rBoNTE(H(c))。
