Specific His-Tag Acylation – A Broad Conjugation Methodology for many new Chemical Biology and Biopharmaceutical Applications.

His-Tag attachment is a common and established procedure for protein purification and many other applications. Jensen et al. developed one more, which is utilizing it for highly specific attachment of linkers or payloads.

Highly selective N-terminal chemical acylation of expressed proteins can be achieved with 4-methoxyphenyl ester as it adds selectively to Gly-His-tags of proteins, while it does not react with epsilon amino functions of surface accessible lysines. Therefore, it expresses a unique possibility to label proteins regio-specifically at the Gly-His-tag position, enabling a wide application for chemical biology and biopharmaceuticals.

4-Methoxyphenyl 2-azidoacetate binds specifically to the N-terminus of the His-tag while other amino functions, e.g., from lysine side chains remain untouched.

General Conjugation Protocol:

A 35 μM solution of GH6-protein in 200 mM HEPES buffer at pH 7.5 is incubated with 40 equiv. of azidoacetyl 4-methoxyphenyl ester for 24 h at 4 °C. The formation of the mono-functionalized product can be observed by ESI-MS and can reach 70% to 90% conversion. A higher conversion rate can be achieved by the addition of two aliquots of 10 equiv. of the acylating agent in the next two days.

Imidazole rings of neighboring histidines in a His-tag catalyze acylation of the N-terminus of glycine via a base catalyzed mechanism.

Mechanistic studies indicate that the very high selectivity of the His-tag acylation is based on specific base catalysis, in which a His side-chain assists deprotonation during the direct acylation of the Gly α-amine (Fig. 25). The ester reacts preferentially with assistance from His side-chain imidazoles since they are not protonated (pKa ~ 6.0) at the pH of the reaction, in contrast to the N-terminal α-amine (pKa ~ 7.6-8.0) and Lys side-chains (pKa ~ 10.5). The presence of the additional five His residues in the His-tag may serve to modulate the basicity of the imidazole nitrogen of the catalytic residue. A recent study has shown that the pKa values of individual His side-chains in a His6-tag span a range from 4.8-7.5.

➔ Please also see the Linkerology section on our webpage.


Reference:
Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence; M. C. Martos-Maldonado, C. T. Hjuler, K. K. Sorensen, M. B. Thygesen, J. E. Rasmussen, K. Villadsen, S. R. Midtgaard, S. Kol, S. Schoffelen and K. J. Jensen; Nat Commun 2018; 9: 3307. https://doi.org/10.1038/s41467-018-05695-3