Mining repetitive motifs in intrinsically disordered proteins to engineer coacervate-forming synthetic polypeptides

Intrinsically disordered proteins (IDPs) have been recognized as key drivers of liquid-liquid phase separation, forming biomolecular coacervates that regulate various intracellular processes. This behavior is frequently associated with low-complexity regions enriched in repetitive amino acid motifs found in IDPs. Based on this principle, a bioinformatics-guided platform was developed to identify and engineer consensus repeat sequences capable of driving LLPS in synthetic protein systems. Sequences from the DisProt database were analyzed by categorizing amino acids into four classes according to charge and hydrophilicity. The resulting simplified sequences were aligned across various repeat unit lengths using a one-hot encoding strategy to identify recurrent motifs. The most promising consensus units were translated back into amino acid sequences, recombinantly expressed in E. coli. The engineered proteins were evaluated for their ability to undergo phase separation with selective binding through turbidimetry and partitioning assays, using the parent protein as a control. The results showed that the synthetic constructs exhibited coacervation behavior similar to that of their parent IDPs, confirming that minimal repetitive motifs were sufficient to reproduce phase separation properties. These engineered materials are proposed as a promising platform with selective binding capabilities for the development of programmable biomolecular condensates and bioinspired soft matter systems, with potential applications in intracellular delivery, synthetic biology, and biomaterials design.