Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid–liquid phase separation of cellular biomolecules

Abstract

The development of countercurrent chromatography (CCC) technology enabled us to achieve higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol and inorganic salts (or dextrans). By eliminating the solid support matrix, all analytes can be recovered from the coiled column after the separation is completed.Recently, it has been found that droplets of biomolecules formed by liquid-liquid phase separation in cells closely relate to the transcription, regulation of signal transduction, and formation of amyloids. Meanwhile, although CCC is a separation technique based on liquid-liquid partitioning of analytes between two immiscible phases, the mechanism of separation could suggest some idea concerning the formation of biomolecule droplets in cells. This article describes the recent advances in the CCC apparatus, the coiled separation column, the choice of a suitable two-phase solvent system, and the application to separation and purification of bioactive macromolecules such as proteins and enzymes, and also discusses the possibility of CCC as a tool to reveal new mechanical roles of biomolecule droplets in the cellular environments.aqueous two-phase solvent system, cellular biomolecules, coil planet centrifuge, coil satellite centrifuge, countercurrent chromatography 1 | INTRODUCTION Recently, it has been found that some proteins in cells function as aggregates after forming droplets in the liquid-liquid phase separation (LLPS) process. This discovery closely relates to various biological behaviors, including transcription, regulation of signal transduction, and formation of amyloids. The transcription factor forms droplets congregated by an electrostatic interaction between the intrinsically disordered domain and DNA and transferred into a higher state of transcription activity (Chong et al., 2018). The study on signal transduction mechanism shows that biomacromolecules connect with one another by weak interaction, and in proteins the intrinsically disordered regions generally relate to their interactions. Stress granules formed temporally in cytoplasm regulate stress response signals by holding various transduction molecules in themselves (Riggs et al., 2020). Furthermore, amyloid, protein aggregate often formed by the failure of folding, is attributed to serious diseases, that is, neurodegenerative diseases such as Alzheimer's disease, Huntington's

Recent trends in multidimensional countercurrent chromatography

“…One of the phases is often a nonpolar organic solvent, such as hexane (Hex) or heptane, while the other is a more polar solvent, such as water or methanol [15]. The two phases are mixed in a countercurrent flow through interconnected chambers typically formed by interconnected coils or columns [16,17]. During MDCCC research, two main approaches exist to heighten peak capacity and enhance resolution.…”