Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion.
Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion.
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通过优化蛋白质分泌的培养条件，最大限度地减少毕赤酵母生物反应器培养中分泌的二价抗人 T 细胞免疫毒素的聚集。
DOI：
10.1016/j.jbiotec.2005.07.004
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发表时间：
2006
影响因子：
4.1
通讯作者：
NevilleJr,DavidM
中科院分区：
文献类型：
--
作者：
Woo,JungHee;Liu,YuanYi;NevilleJr,DavidM
在分泌二价免疫毒素的基因工程毕赤酵母的生物反应器培养物中，64%的分泌免疫毒素以聚集体形式存在，这导致生物活性丧失。对分泌的免疫毒素的生化分析和使用纯化的单体免疫毒素的体外聚集研究表明聚集主要是细胞外事件。通过采用每 10l 初始培养基 0.75mlmin−1 的有限甲醇进料量，减少了耗氧量，从而使生物反应器的搅拌速度从 800 rpm 降低至 400rpm。通过将消泡剂增加到0.6mll−1，空气/液体界面泡沫层的厚度减少了80%。这些步骤将免疫毒素聚集物从 64% 减少到 5%。因此免疫毒素纯化率从 53.0% 增加到 73.8%。同时，在甲醇诱导 163 小时时，该方法在抗毒素生产菌株中将免疫毒素分泌增强至 120mgl−1。我们得出的结论是，最小化剪切力和减少空气/液体界面泡沫面积是减少酵母生物反应器培养物分泌表达时疏水蛋白聚集的关键因素。
In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75mlmin−1per 10l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400rpm. By increasing the anti-foam reagent to 0.6mll−1, the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120mgl−1at 163h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.