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Review
. 2022 Aug 17;144(32):14404-14419.
doi: 10.1021/jacs.2c00129. Epub 2022 Jul 31.

Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation

Ross J Taylor  1 , Michael B Geeson  1 , Toby Journeaux  1 , Gonçalo J L Bernardes  1   2
Affiliations
Review

Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation

Ross J Taylor et al. J Am Chem Soc. .

Abstract

Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biological activity. This perspective focuses on chemical and enzymatic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chemical modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymatic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges associated with using chemical and enzymatic approaches are highlighted and areas requiring further development are discussed.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Overview of methods used to prepare unnatural protein–protein conjugates in the context of all available pathways.
Figure 2
Figure 2
Strategies for preparing protein–protein conjugates using homobifunctional linkers.
Figure 3
Figure 3
Role of opposing charges in preparation of protein–protein conjugates.
Figure 4
Figure 4
Production of protetin–protein conjugates by installing click groups on single cysteine residues.
Figure 5
Figure 5
Production of protein–protein–dye conjugates using orthogonal click chemistry and disulfide rebridging.
Figure 6
Figure 6
Use of vinylphosphonite and Pd-protein OAC heterobifunctional reagents for sequential protein conjugation through cysteine residues.
Figure 7
Figure 7
Methods for preparing protein–protein conjugates using lysine conjugation. DTT: Dithiothreitol. TCEP: tris(2-carboxyethyl)phosphine.
Figure 8
Figure 8
Incorporation of an unnatural amino acid (UAA) enables the preparation of IgG-PE24, a protein–protein heterodimer that is not accessible via a fusion protein pathway.
Figure 9
Figure 9
Sortase-based approach for preparing protein–protein conjugates.
Figure 10
Figure 10
Tyrosine-based approached for protein–protein conjugation.
Figure 11
Figure 11
Enzymatic approaches to install click chemistry functional groups for protein–protein conjugation.
Figure 12
Figure 12
Design of SpyCatcher/SpyTag system and application to preparation of bispecific antibodies.
See this image and copyright information in PMC

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