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

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 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 un-activated  labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibriums to favor product formation which greatly enhances the reaction efficiency. Subsequent optimization 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.

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