We demonstrate that a metastable liquid–liquid phase separation (LLPS) in protein aqueous solutions can be induced by multivalent metal ions at room temperature.We determine the salt and protein partitioning in the two coexisting phases. The structure factor obtained by small angle X-ray scattering provides direct evidence for a short-ranged attraction, which leads to the metastability of the LLPS. An extended phase diagram with three control parameters (temperature, protein and salt concentration) provides a conclusive physical picture consistent with a criterion for the second virial coefficient. The presented isothermal control mechanism of the phase behavior opens new perspectives for the understanding of controlled phase behavior in nature. Furthermore, we discuss the application of this framework in predicting and optimizing condi- tions for protein crystallization.
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