Generating Ultra‐Fast Protein trans ‐Splicing of a Cysteine‐Less and Semisynthetic Split Intein for Chemical Protein Labeling

Cysteine‐less split inteins have recently emerged as valuable addition to the protein labeling and modification toolbox as they can perform the protein trans ‐splicing (PTS) reaction under oxidizing conditions. Furthermore, their use is compatible with the chemical labeling and different redox states of cysteines in the extein sequences and hence the protein of interest. However, a rapidly splicing cysteine‐less split intein with one short precursor fragment easily amenable to solid‐phase peptide synthesis was still missing. A chemically synthesized split intein precursor allows for semisynthetic PTS, attractive to introduce fully synthetic sequence segments into the protein of interest. Here, we generate an optimized variant of the highly efficient split CL (cysteine‐less) intein that splices with an ultra‐fast rate on the second scale, comparable to the best performing split inteins. We achieved the nine‐ to 16‐fold increase of the reaction rate by optimizing the artificial split site and the immediately flanking extein residues. Following chemical synthesis of the N‐terminal precursor with an intein fragment of only 26 amino acids, we demonstrated protein semisynthesis by transferring a short fluorescently labeled peptide tag to a protein's N terminus with ultra‐fast kinetics. The optimized split CL intein will thus further expand chemical protein labeling approaches.