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Protease\u2010cleavable affinity tags are commonly used to improve yield and purity of recombinant proteins, but tag removal adds labor and complexity. Previous attempts to use a selective protease to elute cleaved fusion protein during affinity chromatography have suffered from poor digestion efficiency that greatly diminishes yield, or protease contamination in the eluate. Here, we describe a novel SUMO protease construct that supports rapid, high\u2010yield protease elution. This is the keystone of an end\u2010to\u2010end DNA\u2010to\u2010protein workflow that we optimized for speed, parallelizability, and generalizability. While our workflow is intended for an automated liquid\u2010handler, it can easily be performed manually, making it broadly accessible. We demonstrate the method by producing and characterizing 96 disparate proteins derived from mesophilic organisms. 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