A User's Guide for Phase Separation Assays with Purified Proteins

Abstract

The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.

Keywords: Sup35; low-complexity proteins; membrane-less compartment; membrane-less organelle; phase separation; prion-like protein; protein purification.

Graphical abstract

Fig. 1

Domain structure, net charge, and disorder tendency of phase-separating proteins. The isoelectric point (pI) for each protein is shown after the protein name. CC, coiled-coil; Nter, N-terminal domain; DD, dimerization domain; ZnF, zinc finger; DEXDc, DEAD-like helicases superfamily; HELICc, helicase superfamily; N, N domain; M, middle domain; C, C domain; PLD, prion-like domain; RRM, RNA recognition motif. The net charge was plotted using a sliding window of 20 amino acids. The disorder tendency was predicted by IUPred (see Experimental procedures).

Fig. 2

Constructs used to express Sup35. “3C” denotes a 3C PreScission protease cleavage site, “6 × His Tag” is a 6 × His tag for affinity purification, and “MBP” stands for maltose-binding protein that was used as an affinity tag and for solubilizing the fusion protein. Sup35 was also expressed as a fluorescence proteins fusion (Fluoro), such as monomeric sfGFP, TagRFP, or SnapTag.

Fig. 3

Scratches or air bubbles promote a liquid-to-solid transition of FUS. (a) Effect of scratches on liquid-to-solid transitions of FUS. The bar represents 10 μm. (b) Effect of air bubbles on liquid-to-solid transition of FUS. The bar represents 10 μm.

Fig. 4

Constructs used for expression of human prion-like proteins. “3C” represents a PreScission protease cleavage site, and “TEV” represents a TEV protease cleavage site. The MBP tag was used to improve the expression and solubility of the prion-like proteins. To increase the binding to the affinity column, the 6 × His tag was used. After cleavage by the PreScission protease, one terminal of the PLD was released from the tag. It has been noticed that for some prion-like proteins, such as FET proteins, phase separation can be suppressed when one terminal of the PLD is not released.

Fig. 5

Imaging chamber generated by attaching coverslip to the glass slide using double-sided tape placed parallel to each other, such that a rectangular chamber with two openings on opposite sides is made. The slide and coverslip are shown in gray, and double-sided tapes are shown in blue.