Protocol for the Preparation of a Recombinant Treacle Fragment for Liquid-Liquid Phase Separation (LLPS) Assays.

Liquid-liquid phase separation (LLPS) underlies the spatial organization of the nucleolus, a membraneless organelle responsible for ribosomal RNA (rRNA) transcription and ribosome subunit assembly. One of the key proteins involved in the formation of the fibrillar center of the nucleolus is the treacle, an intrinsically disordered protein that contains low-complexity repeats enriched in charged amino acid residues. In this work, we present a detailed protocol for the bacterial expression and purification of a recombinant fragment of treacle comprising two tandem low-complexity repeat (LCR) modules, with a total length of 136 amino acids. This fragment is intended for subsequent in vitro investigation of its ability to undergo LLPS. The described method enables the production of a soluble, biochemically pure protein preparation suitable for studying the mechanisms of spontaneous condensate formation in a cell-free system. This approach allows for the controlled modeling and quantitative evaluation of the contribution of low-complexity sequences to the phase behavior of treacle, independently of its interactions with cellular partners in vivo. Key features • Protocol describes the expression and purification of a soluble treacle fragment containing two LCR motifs, suitable for in vitro LLPS studies. • Avoids complications associated with full-length treacle expression by using a minimal, well-behaved construct. • Produces biochemically pure protein compatible with phase separation assays under controlled buffer and crowding conditions. • Applicable for dissecting sequence features driving electrostatically mediated LLPS in nucleolar IDR/LCR proteins.