The Numacut TEV protease is an improved variant of the Tobacco Etch Virus (TEV) protease with broadened substrate tolerance in comparison to the wild type enzyme. Evolved by artificial intelligence and directed evolution approaches, the recognition site of the TEV protease (ENLYFQ/S) was broadened to ENLYFQ/X (X: any amino acid, except P). Fusion proteins featuring the ENLYFQ sequence are efficiently cleaved between Q and X releasing N-terminal native target peptides or proteins.