"Tag and Modify" Protein Conjugation with Dynamic Covalent Chemistry

Area of Science:

• Bioconjugation Chemistry
• Chemical Biology
• Protein Engineering

Background:

• Small protein tags are crucial for applications in cellular environments, including controlled drug delivery and dynamic protein complex construction.
• Bioorthogonal, stable, reversible, and biocompatible chemical strategies are needed for effective protein modification and conjugation.
• Dynamic covalent chemistry offers unique opportunities for reversible molecular assembly and modification.

Purpose of the Study:

• To report the first application of dynamic covalent chemistry for the purification and reversible assembly of protein conjugates.
• To demonstrate the utility of boronic acid-based interactions with diols and salicylhydroxamates for protein modification.
• To establish a site-selective method for incorporating boronic acid tags into proteins.

Main Methods:

• Site-selective incorporation of a boronic acid (BA) tag into a model protein (lysozyme) using a disulfide rebridging strategy.
• Purification of the BA-tagged protein via carbohydrate-based column chromatography.
• Dynamic covalent bioconjugation of the BA-tagged protein with a salicylhydroxamate-modified fluorescent dye (BODIPY FL).

Main Results:

• Successful site-selective modification of lysozyme with a boronic acid tag.
• Efficient purification of the tagged protein using carbohydrate affinity chromatography.
• Reversible and "click-like" conjugation of the protein with a fluorescent dye, preserving enzymatic activity.

Conclusions:

• Dynamic covalent chemistry, specifically using boronic acid interactions, provides a versatile platform for protein purification and reversible bioconjugation.
• The developed method allows for site-selective protein tagging and functionalization while maintaining protein integrity and activity.
• This approach holds significant promise for developing advanced protein-based therapeutics and research tools.