Phase Separation and Protein Partitioning in Compartmentalized Cell-Free Expression Reactions

Shuzo Kato, David Garenne, Vincent Noireaux, Yusuke T. Maeda

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


Liquid-liquid phase separation (LLPS) is important to control a wide range of reactions from gene expression to protein degradation in a cell-sized space. To bring a better understanding of the compatibility of such phase-separated structures with protein synthesis, we study emergent LLPS in a cell-free transcription-translation (TXTL) reaction. When the TXTL reaction composed of many proteins is concentrated, the uniformly mixed state becomes unstable, and membrane-less phases form spontaneously. This LLPS droplet formation is induced when the TXTL reaction is enclosed in water-in-oil emulsion droplets, in which water evaporates from the surface. As the emulsion droplets shrink, smaller LLPS droplets appear inside the emulsion droplets and coalesce into large phase-separated domains that partition the localization of synthesized reporter proteins. The presence of PEG in the TXTL reaction is important not only for versatile cell-free protein synthesis but also for the formation of two large domains capable of protein partitioning. Our results may shed light on the dynamic interplay of LLPS formation and cell-free protein synthesis toward the construction of synthetic organelles.

Original languageEnglish (US)
Pages (from-to)3451-3459
Number of pages9
Issue number8
StatePublished - Aug 9 2021

Bibliographical note

Funding Information:
We thank R. Sakamoto and T. Fukuyama for discussion and M. Miyazaki for multipoint measurement in confocal microscopy. This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas JP18H05427 and Scientific Research (B) JP20H01872 (to Y.T.M.), HFSP Research Grant RGP0037/2015 (to V.N. and Y.T.M.), and NSF grant EF1934496 (to V.N.).

Publisher Copyright:
© 2021 American Chemical Society.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't


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