The commercial success of artificial spider silk has been limited by inefficient protein production and costly purification methods. Here, we present a strategy addressing both challenges by combining protein engineering with a purification method based on liquid–liquid phase separation. The engineered spider silk proteins are efficiently produced in microorganisms, while purification costs, processing time, and water consumption are reduced by up to 99%, 80%, and 92%, respectively, compared with conventional chromatography, without requiring tag removal, heat, or denaturing agents. The purified proteins can be directly wet-spun into tough artificial silk fibers using an all-aqueous process. These advances demonstrate the potential of liquid–liquid phase separation for sustainable protein production and could facilitate industrial manufacturing of artificial spider silk.
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