Phase separation and protein partitioning in compartmentalized cell-free expression reactions

TL;DR

This study investigates how liquid-liquid phase separation occurs in cell-free protein synthesis systems, revealing how emulsion conditions and PEG influence phase formation and protein partitioning, which could inform synthetic organelle design.

Contribution

It demonstrates that LLPS can spontaneously occur in cell-free reactions within emulsion droplets and shows how PEG affects phase separation and protein localization.

Findings

LLPS occurs spontaneously in concentrated TXTL reactions within emulsion droplets.

PEG promotes formation of large phase-separated domains capable of protein partitioning.

Emulsion droplet size influences the emergence and coalescence of LLPS droplets.

Abstract

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.