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Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

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Abstract

Asparaginyl endopeptidases (AEPs) are ideal for peptide and protein labeling. However, because of the reaction reversibility, a large excess of labels or backbone modified substrates are needed. In turn, simple and cheap reagents can be used to label N-terminal cysteine, but its availability inherently limits the potential applications. Aiming to address these issues, we have created a chemo-enzymatic labeling system that exploits the substrate promiscuity of AEP with the facile chemical reaction between N-terminal cysteine and 2-formyl phenylboronic acid (FPBA). In this approach, AEP is used to ligate polypeptides with a Asn–Cys–Leu recognition sequence with counterparts possessing an N-terminal Gly–Leu. Instead of being a labeling reagent, the commercially available FPBA serves as a scavenger converting the byproduct Cys–Leu into an inert thiazolidine derivative. This consequently drives the AEP labeling reaction forward to product formation with a lower ratio of label to protein substrate. 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-specifically labeling the N- or C-termini of various proteins.

Graphical abstract: Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

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Supplementary files

Article information


Submitted
08 Apr 2020
Accepted
11 May 2020
First published
12 May 2020

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2020, Advance Article
Article type
Edge Article

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

T. M. S. Tang, D. Cardella, A. J. Lander, X. Li, J. S. Escudero, Y. Tsai and L. Y. P. Luk, Chem. Sci., 2020, Advance Article , DOI: 10.1039/D0SC02023K

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