FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells
FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells
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DOI：
10.1038/s41598-020-66927-5
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发表时间：
2020-06-23
影响因子：
4.6
通讯作者：
Kolmar, Harald
中科院分区：
文献类型：
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作者：
Yanakieva, Desislava;Elter, Adrian;Kolmar, Harald
在这项研究中，我们提出了一种基于功能细胞的简单筛选方法，将分泌酵母细胞和报告哺乳动物细胞共同封装在数百万个含琼脂糖的微滴中。我们的系统与利用标准荧光激活细胞分选仪 (FACS) 的超高通量选择兼容，无需进行广泛的调整和优化。在一项模型研究中，我们将分泌鼠白细胞介素 3 (mIL-3) 的酿酒酵母细胞与鼠 Ba/F3 报告细胞共同封装，这些细胞在受到 mIL-3 刺激后表达绿色荧光蛋白 (GFP)，并且与分泌非功能性 mIL-3 突变体的酵母细胞的对照相比，可以观察到荧光信号的特异性和强诱导。我们证明通过两个连续的共封装和 FACS 循环，可以成功富集表达无活性细胞因子变体的细胞中 1:10,000 稀释的激活 mIL-3 wt 分泌酵母细胞。这表明所提出的策略适用于基于高多样性酵母的文库的功能筛选，并证明了其有效分离分泌生物活性重组蛋白的克隆的潜力。
In this study, we present a straightforward approach for functional cell-based screening by co-encapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarose-containing microdroplets. Our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and FACS. This indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins.