TY  - JOUR
TI  - Protocol for the Preparation of a Recombinant Treacle Fragment for Liquid–Liquid Phase Separation (LLPS) Assays
AU  - Petrova, Nadezhda V.
AU  - Balagurov, Konstantin I.
AU  - Razin, Sergey V.
AU  - Velichko, Artem K.
VL  - 15
IS  - 18
PY  - 2025
DA  - 2025/09/20
SP  - e5439
C1  - Bio-protocol 2025;15:e5439
DO  - 10.21769/BioProtoc.5439
UR  - https://doi.org/10.21769/BioProtoc.5439
AB  - 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.
KW  - Treacle
KW  - Liquid–liquid phase separation (LLPS)
KW  - Biomolecular condensates
KW  - Nucleolus
KW  - Low-complexity repeat (LCR)
KW  - Peptide purification
KW  - Intrinsically disordered protein
KW  - Bacterial expression
KW  - Maltose-binding protein (MBP) fusion protein
KW  - Tobacco Etch Virus (TEV) protease
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False