The Effect of Protein Tagging on Aggregation and Phase Separation

Protein tags are widely used for purification, solubilization, detection, and imaging, yet they can substantially alter protein self-assembly. This interference is particularly significant for intrinsically disordered proteins and low-complexity domains, whose aggregation and phase separation are mediated by weak multivalent interactions that are easily disrupted by exogenous elements. In this review, we examine how affinity tags, solubility tags, fluorescent proteins, and chemical labels influence aggregation, amyloid formation, and liquid-liquid phase separation (LLPS). We first classify recurring perturbation mechanisms into six primary categories: solubility enhancement, artificial multivalency, electrostatic interactions, local effects, metal coordination, and positional dependence. Crucially, these non-exclusive mechanisms often operate simultaneously within a single construct. We then review representative case studies across pathogenic amyloids, RNA-binding proteins, viral inclusions, functional amyloids, yeast prions, and membrane proteins. These examples demonstrate that tags alter assembly kinetics, phase boundaries, material properties, fibril morphology, oligomeric states, and observed phenotypes, rather than merely serving as neutral tools for detection. In some systems, tags suppress intrinsic assembly; in others, they promote non-native condensation or stabilize alternative aggregate states. Finally, we discuss practical experimental strategies to distinguish intrinsic protein behavior from construct-dependent effects, emphasizing matched comparisons, orthogonal validation, and the careful interpretation of measurements based on tag cleavage or fluorescence. Collectively, the evidence indicates that protein tags should be treated as experimental variables that shape assembly states rather than as inert technical additions.