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

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.

Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer

“…Furthermore, we hope we have provided a method and proof-of-concept for the generation of such three-protein constructs to enable further innovation in the field. We also hope we have demonstrated the power of bioorthogonal chemical strategies for protein-protein conjugation, as while this area of research has been gaining momentum recently, 5,6 there is much untapped potential that is still waiting to be uncovered.…”

“…For a more comprehensive overview of the subject of chemical bsAbsynthesis, the readers are referred to two recent reviews on the topic. 5,6 The most modern purely chemical approaches for site-selective homogeneous bsAb formation rely on re-bridging the solvent accessible interchain disulfide bonds of antibodies or their antigen-binding fragments (Fabs). As the natural abundance of cysteine is low 7 and most antibodies contain only four readily accessible disulfides, with Fabs containing only one, site-selective modification of these proteins can be achieved in this way.…”

Disordered Domain Shifts the Conformational Ensemble of the Folded Regulatory Domain of the Multidomain Oncoprotein c-Src

“…All protiated and deuterated proteins were overexpressed and purified from Escherichia coli extracts. Staphylococcus aureus transpeptidase, a pentamutant version of sortase (sortase 5M), was used to enzymatically ligate the purified SH4UD and SH3-SH2 domains for SANS experiments. , The plasmid containing the gene that encodes pentamutant sortaseA was a gift from David Liu (Addgene plasmid # 75144) . Briefly, the optimized conditions for sortase-mediated ligation reaction were as follows: 20 μM SH4UD, 50 μM SH3-SH2, 75 nM sortase 5M; 10 mM CaCl 2 in 50 mM Tris HCl for 16 h at 4 °C.…”

Strategies for Conditional Regulation of Proteins

“…Chemical cross-linking is another strategy to regulate proteins. This is achieved through bioconjugate chemistry, or bioorthogonal reactions or enzymatic labeling as in the case of protein–protein (homo- or hetero-) dimers. − Alternatively, bifunctional molecules like glutaraldehyde or bis-acids (EDC/NHS chemistry) may be used to cross-link proteins on their basic residues like lysine nonspecifically. , The latter approach, though very efficient, may ultimately inactivate the protein. In the context of vaccine design, glutaraldehyde-based cross-linking was used to stabilize the HIV envelope glycoprotein BG505 to improve neutralizing antibodies against HIV …”