Highly selective chemical modification of poly-His tagged peptides and proteins
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.
Original language | English |
---|---|
Title of host publication | Methods in Enzymology |
Number of pages | 29 |
Volume | 698 |
Publisher | Elsevier |
Publication date | 2024 |
Pages | 111-139 |
Chapter | 5 |
DOIs | |
Publication status | Published - 2024 |
Series | Methods in Enzymology |
---|---|
ISSN | 0076-6879 |
Bibliographical note
Funding Information:
We thank all our coauthors on previous papers for their contributions. We thank the VILLUM FONDEN for funding the Biomolecular Nanoscale Engineering Center (BioNEC), a VILLUM center of excellence, grant number VKR022710. The Novo Nordisk Foundation is acknowledged for funding the Center for Biopharmaceuticals and Biobarriers in Drug Delivery (BioDelivery; Grand Challenge Program; NNF16OC0021948), and finally Independent Research Fund Denmark for a grant (2035-00277B) to KJJ.
Publisher Copyright:
© 2024
- Antibodies, Antibody-drug conjugates, Biopharmaceuticals, Chemical protein modification, Fluorescent probes, His tag, Protein conjugates, Selective modification
Research areas
ID: 397903223