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Maximizing recovery of native protein from aggregates by optimizing pressure treatment

机译：通过优化压力处理，最大程度地从聚集体中回收天然蛋白质

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摘要

Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 mug/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.
机译：从聚集体中回收天然蛋白质是重组蛋白质生产中的常见障碍。最近的报告表明，就产量和工艺简便性而言，静水压力是传统变性和稀释技术的一种有吸引力的替代方法。为了确定过程变量的影响，我们对尾钉骨料进行了多种压力处理条件。仅在240 MPa下短时温育（<5分钟）即可获得最大的天然尾穗产量。然而，尽管多次加压循环，一些尾钉聚集体仍能抵抗压力。将后压孵育时间延长至4天，可将聚集体的天然蛋白质产量从19.4 +/- 0.9提升至47.4 +/- 19.6马克杯/毫升（非聚集物质中天然三聚体的产率约为78％）。当与以前的动力学数据结合使用时，在几天的时间范围内，单体几乎完全转化为三聚体，因此可以确定三个后压力动力学相：由结构化二聚体转化为三聚体的快速相（30分钟），一个中间相由单体转化成聚集体的反应（100分钟）和由单体转化成三聚体的慢相反应（天）。优化结构化二聚体的生产可以产生最高水平的折叠蛋白。典型的可折叠添加剂（例如甘油）或低温孵育不会提高产量。

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