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

Author:

Arnott Zoe L. P.12,Morgan Holly E.13ORCID,Hollingsworth Kristian1ORCID,Stevenson Charlotte M. E.1ORCID,Collins Lawrence J.1ORCID,Tamasanu Alexandra1ORCID,Machin Darren C.1ORCID,Dolan Jonathan P.14ORCID,Kamiński Tomasz P.1ORCID,Wildsmith Gemma C.1,Williamson Daniel J.15,Pickles Isabelle B.16ORCID,Warriner Stuart L.1ORCID,Turnbull W. Bruce1ORCID,Webb Michael E.1ORCID

Affiliation:

1. School of Chemistry and Astbury Centre for Structural Molecular Biology University of Leeds Woodhouse Lane Leeds LS2 9JT UK

2. Present address: Centre for Process Innovation, Central Park The Nigel Perry Building, 1 Union St Darlington DL1 1GL United Kingdom

3. Present Address: Ashfield MedComms City Tower, Piccadilly Plaza Manchester M1 4BT United Kingdom

4. Present Address: School of Chemical and Physical Sciences & Centre for Glycoscience Research and Training Keele University Keele, Staffordshire ST5 5BG United Kingdom

5. Present Address: Iksuda Therapeutics The Biosphere Draymans Way Newcastle upon Tyne NE4 5BX United Kingdom

6. Present Address: York Structural Biology Laboratory, Department of Biology University of York Heslington, York YO10 5DD United Kingdom

Abstract

AbstractQuantitative 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.

Funder

Biotechnology and Biological Sciences Research Council

Engineering and Physical Sciences Research Council

Publisher

Wiley

Subject

General Medicine

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