2024
DOI: 10.1002/anie.202310862
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Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Zoe L. P. Arnott,
Holly E. Morgan,
Kristian Hollingsworth
et al.

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 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 e… Show more

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Cited by 6 publications
(2 citation statements)
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“…Taking into account the recent study on biotinylating two model nanobodies by sortagging with the peptidic substrate GGGK-biotin, we can confirm the efficiency and convenience of this technique by extending it toward the use of various nonpeptidic amine nucleophiles and diverse nanobody substrates. While the reversibility of reactions and the associated yield and side-product issues continue triggering significant endeavors to improve sortagging methodology, the efficiency of biotinylation with the GGGK-biotin substrate we have observed in this study is similar to the nonpeptidic biotin derivatives. However, our experiments were done in the optimized conditions strongly favoring aminolysis versus hydrolysis processing, such as large excess of incoming nucleophiles, elevated buffer pH, and lowered temperatures, and also with a single type of protein substrate (nanobodies) and labeling payload residue (biotin).…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…Taking into account the recent study on biotinylating two model nanobodies by sortagging with the peptidic substrate GGGK-biotin, we can confirm the efficiency and convenience of this technique by extending it toward the use of various nonpeptidic amine nucleophiles and diverse nanobody substrates. While the reversibility of reactions and the associated yield and side-product issues continue triggering significant endeavors to improve sortagging methodology, the efficiency of biotinylation with the GGGK-biotin substrate we have observed in this study is similar to the nonpeptidic biotin derivatives. However, our experiments were done in the optimized conditions strongly favoring aminolysis versus hydrolysis processing, such as large excess of incoming nucleophiles, elevated buffer pH, and lowered temperatures, and also with a single type of protein substrate (nanobodies) and labeling payload residue (biotin).…”
Section: Resultsmentioning
confidence: 99%
“…This often leads to low reactions yields, the necessity of using large excess of incoming nucleophiles to drive the reaction equilibrium toward a target conjugate formation, and an irreversible accumulation of nonfunctionalized byproduct of hydrolysis of the acceptor protein-enzyme intermediate, which is cumbersome to separate from the desired conjugate . A large number of ingenious approaches aimed at resolving these issues have been reported, including the extensive engineering of sortases, as recently reviewed, , use of immobilized sortases, , enhancement of enzyme–substrate proximity, inactivation or removal of reaction products, and some others. , Substrate improvement through knowledge-based use of synthetic nucleophiles of nonpeptidic nature may potentially serve as a magic bullet for the ultimate optimization of C-terminal protein sortagging. Synthetic amines may potentially circumvent the limitations of peptide-based nucleophiles by being more efficient sortase substrates, by suppressing sortase reaction reversibility, as well as by allowing much greater versatility in experimental designs of protein functionalization.…”
Section: Introductionmentioning
confidence: 99%