Semisynthesis of cytotoxic proteins using a modified protein splicing element

Abstract Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI) , were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C‐terminus of a recombinant protein. Nucleophilic attack on this thioester by the N‐terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI , respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol‐induced cleavage of the precursor led to the liberation of the target protein with a C‐terminal thioester‐tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full‐length products that displayed catalytic activity indicative of the wild‐type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8. 2 s‐1 and 1. 5 mM, in good agreement with reported values of 8. 3 s‐1 and 1. 2 mM (Hodges&Merrifield, 1975). Ligated HpaI had a specific activity of 0. 5–1. 5 × 106 U/mg, which compared favorably with the expected value of 1–2 × 106 U/mg (J. Benner, unpubl. obs. ). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.