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His Tag Acylation

Go to the profile of Knud Jorgen Jensen
Aug 20, 2018
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The paper in Nature Communications is here: go.nature.com/2wgrCH9

Chemical modifications of proteins to introduce new, non-natural functions are of great importance in many areas. This includes fluorescently tagged proteins as probes for chemical biology, and biopharmaceutical conjugates, such as PEGylated or lipidated proteins, or antibody-drug conjugates. However, it is important that the chemical modification is introduced at a single site in the protein, otherwise mixtures of protein conjugates will be obtained.

Selective chemistry on proteins remains a huge challenge. There are only few reactions that are used widely. Many reactions have been explored but few have been adapted for general use.

Some years ago one of us (KJJ) was at a project meeting at the University of Copenhagen (Vernet et al, Molecular Pharmaceutics, 2016, 13(5), 1587-1598) when a colleague mentioned the annoying N-terminal side-reaction, gluconoylation, that had modified his protein. This side-reaction was well-described and Geoghegan and coworkers had shown that this side-reaction only occured at the N-terminus (Geoghegan et al, Analytical Biochemistry, 1999, 267, 169-184). We envisioned that this undesirable side-reaction could be optimized into a selective, hence desirable way to modify proteins:

‘A side-reaction could be optimized to a desired main reaction.’

This then became the project for a master thesis (CTH) and the first outline of the optimization was achieved. We realized that the best yields for this N-terminal acylation with gluconolactone required an N-terminal Gly directly followed by the poly-His sequence. Ultimately, we identified GlyHis6 as the preferred N-terminal sequence. We call this a ‘His acylation tag’. It is thus not a conventional His tag, but requires an N-terminal GlyHis6 with no additional N-terminal extension. It is essential that the Gly is the N-terminal amino acid. However, it can still be used for purification, like a conventional His tag.

The next crucial step was the discovery that phenyl esters, in particular 4-methoxyphenyl esters (MCMM), could be used as highly selective acylating agents instead of gluconolactone. We then moved on to study proteins with the N-terminal GlyHis6. We were joined by an experienced protein chemist (SS) and then demonstrated this method for more complicated proteins.

His tag acylation uses catalysis by imidazoles in proximity. The catalytic effect of imidazoles on hydrolysis had already been studied in the 50’s and 60’s. Our own mechanistic studies (MBT) have shown that the catalytic effect of the His tag acylation likely operates via facilitating proton abstraction in the acylation reaction.

Our His acylation tag only works at the N-terminal end and after acylation, the His acylation tag remains in the protein. However, considering how widely used conventional N-terminal His tags are in proteins science, we hope that our His acylation tag will be used widely for selective protein labeling and other modifications.

Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence

Manuel C. Martos-Maldonado, Christian T. Hjuler, Kasper K. Sørensen, Mikkel B. Thygesen, Jakob E. Rasmussen, Klaus Villadsen, Søren R. Midtgaard, Stefan Kol, Sanne Schoffelen, Knud J. Jensen

https://rdcu.be/4FCj

Go to the profile of Knud Jorgen Jensen

Knud Jorgen Jensen

Professor, University of Copenhagen

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