Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d ‐Aminopeptidase

Arnott, Zoe L. P.; Morgan, Holly E.; Hollingsworth, Kristian; Stevenson, Charlotte M. E.; Collins, Lawrence J.; Tamasanu, Alexandra; Machin, Darren C.; Dolan, Jonathan P.; Kamiński, Tomasz P.; Wildsmith, Gemma C.; Williamson, Daniel J.; Pickles, Isabelle B.; Warriner, Stuart L.; Turnbull, W. Bruce; Webb, Michael E.

Abstract

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N‐terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Citation

Arnott, Z. L. P., Morgan, H. E., Hollingsworth, K., Stevenson, C. M. E., Collins, L. J., Tamasanu, A., …Webb, M. E. (2024). Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d ‐Aminopeptidase. Angewandte Chemie, Article e202310862. https://doi.org/10.1002/ange.202310862

• Journal Article Type: Article
• Acceptance Date: Dec 10, 2023
• Online Publication Date: Dec 10, 2023
• Publication Date: Jan 11, 2024
• Deposit Date: Jan 16, 2024
• Journal: Angewandte Chemie
• Print ISSN: 0044-8249
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Article Number: e202310862
• DOI: https://doi.org/10.1002/ange.202310862
• Keywords: Protein Labelling, Transpeptidase, Sortase, Coupling Enzymes