Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain

The removal of N-terminal methionine (Met) or fused N-terminal purification tags to expose the first amino acid of the target protein is critically important for studying the potential regulatory role of the N-terminal residue. However, current tag-removal approaches typically rely on enzymatic cleavage or require harsh reaction conditions. Here, we report a strategy for expressing proteins of interest (POI) with custom N-terminal amino acids by introducing an engineered cysteine protease domain (CPD) tag at the N-terminus. The cleavable tag is chemically triggered by inositol hexakisphosphate (InsP₆), enabling precise generation of proteins with a user-defined N-terminus. Through systemic design and engineering of the N-terminal CPD tag, we successfully achieved POI variants with N-terminal Gly, Ser, His, Lys and other residues except Pro. In addition to the model protein, a Her2-targeting nanobody, we also successfully produced a RNF43-specific nanobody with an N-terminal Gln (N-Gln), an EGFR-targeting nanobody with N-Ala, the Sortase A enzyme with N-Gln, the fluorescent protein TurboGFP with N-Glu, and the sialic acid transferase Δ15 Pd2,6ST with N-Cys. The construction of engineered nCPD-His10-POI further produced POI variants with a customized N-terminus and without any purified tags. Overall, the established approach enables high-yield protein expression and enzyme-independent, single-step removal of the redundant tag to yield proteins with the desired N-terminal residues, offering a valuable option for investigating N-terminal modifications and their functional implications.