Highly Selective Lysine Acylation in Proteins Using a Lys‐His Tag Sequence

Abstract

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N‐terminal Gly‐His6 segment. This tag promoted acylation of the N‐terminal Nα‐amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn‐Lys‐Hism, which we term Lys‐His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ‐amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys‐His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C‐terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys‐His tag acylation method.

Selective Acylation of Proteins at Gly and Lys in His Tags

“…The method was shown to facilitate the modification of the antibody Rituximab (Figure 4). Finally, we demonstrated that the reaction was selective towards a Lys‐His tagged protein when present in a solution of various other, non‐tagged proteins [35] …”

“…Finally, we demonstrated that the reaction was selective towards a Lys-His tagged protein when present in a solution of various other, non-tagged proteins. [35] Figure 5. Possible mechanisms for the autocatalytic, selective acylation of Gly-and Lys-His tags.…”

One-Step Conversion of NHS Esters to Reagents for Site-Directed Labeling of IgG Antibodies

“…For both of the control probes insignificant labeling is observed, making 2b and 2k promising reagents for site-directed labeling. Especially for 2b this was expected, as esters containing the p -methoxyphenol motive in 2b have been reported previously for protein labeling, where they showed negligible global labeling. , Moving forward, we choose to focus on the motive from 2b , as this phenol showed complete activation in our screen, furthermore solubility issues in aqueous buffer had been observed for 2k .…”

“…methoxyphenol motive in 2b have been reported previously for protein labeling, where they showed negligible global labeling. 32,33 Moving forward, we choose to focus on the motive from 2b, as this phenol showed complete activation in our screen, furthermore solubility issues in aqueous buffer had been observed for 2k.…”

Enhancing activity of food protein‐derived peptides: An overview of pretreatment, preparation, and modification methods

“…Based on the modified residue's location, acylation can be classified into three types: N‐acylation, O‐acylation, and C‐acylation (Figure 4a). N‐acylation refers to the attachment of the acyl group to the amino group of the N‐terminal residue of the peptide (Kofoed et al., 2022). O‐acylation entails the attachment of the acyl group to the oxygen atom of a serine or threonine residue within the peptide (Wiktorowicz et al., 2012).…”