Engineering Design of Artificial Phase-Separating Proteins.

Phase-separating proteins are evolutionarily conserved biomolecules that orchestrate key cellular processes. Although the biophysical principles underlying liquid-liquid phase separation (LLPS)-including multivalent interactions, intrinsically disordered regions (IDRs), and the sticker-spacer framework-are now well established, translating these insights into actionable engineering guidelines remains open challenges. Here, we present a design-oriented synthesis for engineering artificial phase-separating proteins. Drawing on a curated dataset, we characterize the physicochemical patterns associated with LLPS behavior, we evaluate current computational predictors, revealing systematic blind spots in hydrophobicity-rich or modular repeat architectures. Building on these insights, we propose modular design heuristics that encompasses sequence-level composition and patterning, valency control via repeat architecture, and topological fusion strategies including terminal tags, telechelic constructs, and interleaved block architectures. These design levers are mapped onto applications such as protein purification, catalytic condensates, stimuli-responsive systems, and mechanically hydrogels and fibers. Finally, we present an integrated design-build-test-learn (DBTL) framework that bridges predictive modeling, experimental iteration, and functional validation. By treating phase separation as an engineerable rather than merely emergent property, this review offers both a conceptual map and a practical roadmap for the rational construction of artificial phase-separating proteins across biotechnological and biomedical contexts.