Engineering Subtilisin into a Fluoride-Triggered Processing Protease Useful for One-Step Protein Purification

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Abstract

Subtilisin was engineered into a highly specific, processing protease, and the subtilisin prodomain was coengineered into an optimized recognition sequence. This involved five steps. First, a robust subtilisin mutant was created, which could tolerate the subsequent mutations needed for high specificity. Second, the substrate binding pocket was mutated to increase its sequence selectivity. Third, the subtilisin prodomain was engineered to direct cleavage to the junction of any protein fused to it. Fourth, the active site of subtilisin was engineered to kinetically isolate binding and cleavage reactions. Finally, specific anions were identified to trigger the processing reaction, with fluoride ions being particularly useful. The ability to isolate the binding and processing steps with a triggering mechanism created a protease with a virtual on-off switch. This allowed column-immobilized processing subtilisin to be used as both the affinity ligand and processing protease for one-step purification of proteins. Fusion proteins tagged with the engineered prodomain can be bound to the column and washed free of contaminants. Cleavage can be triggered by the addition of fluoride to release the pure target protein. The column is then regenerated by stripping off the tightly bound prodomain at pH 2.1. Ten proteins have been purified to date by this method.

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