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

TL;DR

Scientists created a fast, cysteine-free protein labeling tool that works under oxidizing conditions and allows precise chemical modification of proteins.

Contribution

The first ultra-fast, cysteine-less split intein with a short N-terminal fragment suitable for chemical synthesis is developed.

Findings

An optimized cysteine-less split intein splices proteins in seconds, matching top-performing split inteins.

The N-terminal fragment (26 amino acids) was chemically synthesized and used to label a protein's N terminus with a fluorescent tag.

The new intein enables semisynthetic protein labeling independent of redox conditions.

Abstract

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.

Protein trans‐splicing (PTS) by split inteins is a powerful approach for protein semisynthesis to generate proteins with site‐specific modifications that are difficult or impossible to introduce biosynthetically. Here, the authors report the first ultra‐fast and cysteine‐less split intein with a sufficiently short N‐terminal fragment amenable to chemical synthesis to carry out semisynthetic PTS independent of redox conditions.© 2026 WILEY‐VCH GmbH

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