School of Biotechnology, Jawaharlal Nehru University, New delhi-67, India.
AMB Express. 2011 Oct 22;1(1):33. doi: 10.1186/2191-0855-1-33.
There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.
需要阐明宿主细胞对重组蛋白合成诱导的代谢应激反应的产物特异性特征。为此，首选方法是转录组谱分析，它可以更深入地了解复杂的全局代谢网络中发生的变化。比较了三种表达不同蛋白质（重组人干扰素-β（rhIFN-β）、木聚糖酶和绿色荧光蛋白（GFP））的分批补料培养物的转录组谱。我们观察到所有三种表达蛋白都存在营养物质摄取和利用途径的抑制。因此，甘油转运蛋白和参与 ATP 合成以及需氧呼吸的基因受到严重下调。另一方面，当可溶性蛋白在不同启动子下表达时，氨基酸摄取和生物合成基因受到显著抑制，但当产物作为包含体（IB）表达时则不受抑制。在 T7 启动子（rhIFN-β和木聚糖酶）下的高水平表达触发了细胞降解机制，如渗透保护剂、蛋白酶和 mRNA 降解基因，它们被高度上调，而在相对较弱的 ara 启动子下 GFP 表达时则没有这种趋势。因此，用于重组蛋白生产的更好宿主平台的设计需要考虑到细胞对蛋白表达的特定反应性质。
