Liu Zhu, Bartlow Patrick, Varakala Rajaramesh, Beitle Robert, Koepsel Richard, Ataai Mohammad M
Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA.
J Chromatogr A. 2009 Mar 20;1216(12):2433-8. doi: 10.1016/j.chroma.2009.01.020. Epub 2009 Jan 15.
After some initial optimization, a downstream process comprised of one or several chromatography steps removes the majority of the host proteins and achieves a reasonable degree of purification. The separation of remaining contaminant proteins from the target protein could become very difficult and costly due to their similar physicochemical properties. In this paper we describe a highly efficient strategy, based on proteomic analysis and elution chromatography, by which a protein of interest may be isolated from copurifying contaminants. Mutant strains of Escherichia coli were prepared that are deficient in three prevalent host proteins found in a strategic fraction of an elution profile of nickel immobilized affinity chromatography. Recombinant green fluorescent protein (GFPuv) served as a model protein and its elution was directed to this optimized fraction with an N-terminus hexahistidine tag (his(6)), thereby easing its recovery. We demonstrate that proteomic data can facilitate the rational engineering of host cell expressing the target protein and the design of an efficient process for its purification.
经过一些初步优化后，由一个或几个色谱步骤组成的下游工艺可去除大部分宿主蛋白，并实现合理程度的纯化。由于剩余的污染物蛋白与目标蛋白具有相似的物理化学性质，因此将它们与目标蛋白分离可能变得非常困难且成本高昂。在本文中，我们描述了一种基于蛋白质组学分析和洗脱色谱的高效策略，通过该策略可以从共纯化的污染物中分离出感兴趣的蛋白质。制备了大肠杆菌突变株，这些突变株缺乏在镍固定亲和色谱洗脱图谱的一个关键部分中发现的三种常见宿主蛋白。重组绿色荧光蛋白（GFPuv）用作模型蛋白，其洗脱被引导至具有N端六组氨酸标签（his(6)）的这个优化部分，从而便于其回收。我们证明蛋白质组学数据可以促进表达目标蛋白的宿主细胞的合理工程改造及其纯化高效工艺的设计。
