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  • 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.

Scientific reports (2020-06-25)
Desislava Yanakieva, Adrian Elter, Jens Bratsch, Karlheinz Friedrich, Stefan Becker, Harald Kolmar
摘要

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.

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Sigma-Aldrich
青链霉素, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
OptiPrep密度梯度培养基, used for cell and subcellular organelle isolation
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琼脂糖, Ultra-low Gelling Temperature