Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are commonly studied in vitro using protease-mediated removal of solubilizing tags to induce condensation under controlled conditions. Tobacco Etch Virus (TEV) protease is widely used for this purpose and is generally assumed to remain soluble and inert during condensate reconstitution. Here, we show that in RNA-containing systems, TEV protease variants can interact with RNA, leading to aggregation and changes in the phase behavior of the target protein. Using confocal microscopy, turbidity measurements, and mass photometry, we demonstrate that commonly used TEV protease variants differ in their propensity to undergo RNA-dependent aggregation. The widely used pRK793 TEV protease forms large RNA-associated aggregates. We further show that RNA-TEV aggregation alters the morphology and organization of protein-RNA condensates formed by well-characterized phase-separating proteins, including PGL-3 and FUS. Together, our findings show that TEV protease can directly impact in vitro LLPS assays through RNA binding and aggregation. These results underscore the importance of validating protease-based induction strategies and incorporating appropriate controls when reconstituting biomolecular condensates, particularly in RNA-rich systems.
Larson, J. A., Iglesias-Fuller, D., Putnam, A. A.
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