Aw Rochelle, Barton Geraint R, Leak David J
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
Centre for Synthetic Biology and Innovation, Imperial College London, London, SW7 2AZ, UK.
Appl Microbiol Biotechnol. 2017 Jun;101(12):5045-5058. doi: 10.1007/s00253-017-8317-2. Epub 2017 May 22.
Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.
克隆变异是毕赤酵母表达系统中一个公认的现象，即从理论上相同的转化子中可观察到分泌蛋白的一系列特定生产力。这意味着需要检测大量的转化子以获得具有代表性的样本，并且在商业蛋白质生产中，公司通常会筛选数千个转化子以选择分泌能力最强的转化子。在此，我们通过对携带人血清白蛋白基因的克隆进行表征，对这一现象进行了详细研究。测定了九个克隆（每个克隆均含有单拷贝人血清白蛋白基因，通过定量PCR鉴定）的滴度，并将这些克隆分为三类，即高、中、低水平分泌者。使用微阵列进行的转录组分析表明，没有调控模式与滴度始终相关，这表明克隆变异的原因是多样的。然而，一些生理变化似乎是滴度差异的基础，这表明高分泌菌株存在不止一种生化特征。一个异常的低分泌菌株显示出高转录水平，似乎处于营养饥饿状态，这进一步强调了克隆变异的复杂性。
