A flow cytometric assay for screening improved heterologous protein secretion in yeast
A flow cytometric assay for screening improved heterologous protein secretion in yeast
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DOI：
10.1021/bp0600233
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发表时间：
2006-08-01
影响因子：
2.9
通讯作者：
Wittrup, K. Dane
中科院分区：
文献类型：
--
作者：
Rakestraw, J. Andy;Baskaran, Angelin R.;Wittrup, K. Dane
选择高产的蛋白质表达宿主是生物过程设计的重要组成部分。作为传统平板、光环和抑制筛选的替代方案，我们描述了一种高通量流式细胞术测定，即细胞表面分泌测定（CeSSA），可用于选择改善的异源蛋白质分泌从群体S。酿酒酵母突变体。配体通过PEG接头共价连接到细胞表面，并且当细胞分泌结合栓系配体的蛋白质时，蛋白质被捕获在表面上，在那里它可以被标记，并且使用流式细胞术分选细胞。本报告描述了三种不同的蛋白质/配体相互作用，已经证明了这个系统。在从野生型分泌子的背景群体中分离3倍高的分泌子中，已经验证了23- 54倍的单程分选富集，使得该系统适用于大文库筛选（10（8）个克隆）。开发了数学模型以进一步改进测定的参数。该模型进行了验证与时间过程的数据，并预测一个最佳的筛选窗口。该模型还预测了上述验证实验的60倍富集率。随着该选择系统的发展，可以克服传统的，特别是基于平板的分泌测定所呈现的限制，使得可以在更接近发酵罐中细胞所经历的生长生理学的条件下检查更大的搜索空间。
Selection of highly productive hosts for protein expression is a significant component of bioprocess design. As an alternative to traditional plate, halo, and suppression-based screens, we describe a high-throughput, flow cytometric assay, the Cell Surface Secretion Assay (CeSSA), that can be used to select for improved heterologous protein secretion from a population of S. cerevisiae mutants. A ligand is covalently attached to the cell surface via a PEG linker, and as cells secrete a protein that binds the tethered ligand, the protein is captured on the surface where it can be labeled and the cells sorted using flow cytometry. This report describes three different protein/ligand interactions that have been demonstrated with this system. Single-pass sorting enrichments from 23- to 54-fold have been validated in the separation of a 3-fold higher secretor from a background population of wild-type secretors making this system applicable to large library screening (10(8) clones). A mathematical model was developed to improve the parameters of the assay further. The model was validated with time course data and predicts an optimal screening window. The model also predicts a 60-fold enrichment rate for the validation experiment described above. With the development of this selection system, limitations presented by traditional, particularly plate-based, secretion assays can be overcome so that a larger search space can be examined under conditions closer to the growth physiology experienced by cells in fermentors.