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Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling

preprint
revised on 12.02.2020 and posted on 13.02.2020 by Simon Tang, Davide Cardella, Alexander J. Lander, Xuefei Li, Yu-Hsuan Tsai, Louis YP Luk
Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (e.g. depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

Funding

the Leverhulme Trust

Wellcome Trust

Royal Society

EPSRC

History

Email Address of Submitting Author

lukly@cardiff.ac.uk

Institution

Cardiff University

Country

United Kingdom

ORCID For Submitting Author

0000-0002-7864-6261

Declaration of Conflict of Interest

There is no conflict of interest

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