Enzymatic Labeling of Proteins: Techniques and Approaches

Abstract

Site-specific modification of proteins is a major challenge in modern chemical biology due to the large number of reactive functional groups typically present in polypeptides. Because of its importance in biology and medicine, the development of methods for site-specific modification of proteins is an area of intense research. Selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally-occurring post-translational modifications, for creating antibody-drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics and protein-protein interactions and for the preparation of protein-polymer conjugates. One of the most important approaches for protein labeling is to incorporate bioorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated tags then enable reactions that are chemoselective, whose functional groups are not only inert in biological media, but also do not occur natively in proteins or other macromolecules. This review article summarizes the enzymatic strategies, which enable site-specific functionalization of proteins with a variety of different functional groups. The enzymes covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase and N-myristoyl transferase.

Programmable polyproteams built using twin peptide superglues

“…In future work, it will be interesting to explore SpyCatcherSnoopCatcher units with alternative sizes and orientations, as well as using other linkage chemistries for solid-phase synthesis. Sortase, subtiligase, transglutaminase, and split inteins enable covalent protein-protein interaction that is either traceless or leaves a short peptide tag (39)(40)(41)(42)(43), and these approaches will be valuable to test in the future with MBPx attachment and maltose elution. However, those reactions pass through a (thio)ester intermediate, so hydrolysis may compete with ligation (39,40).…”

“…Sortase, subtiligase, transglutaminase, and split inteins enable covalent protein-protein interaction that is either traceless or leaves a short peptide tag (39)(40)(41)(42)(43), and these approaches will be valuable to test in the future with MBPx attachment and maltose elution. However, those reactions pass through a (thio)ester intermediate, so hydrolysis may compete with ligation (39,40). Artificial amino acids for bioorthogonal reaction would enable minimal modification but with some issues: the increased complexity of module expression and competing reactions [such as azide reduction (44), alkyne reaction with thiols (45), and spontaneous tetrazine degradation (46)] as well as competition from suppression of stop codons by normal amino acids (47).…”

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

“…Enzymes that recognize a specific amino acid tag, usually ranging from four up to 15 residues [117,118] can be exploited for site-specific antibody conjugation. The uniqueness of the tag prevents random modification and allows the site of modification to be exactly determined in a similar manner as the incorporation of additional cysteines or NCAA.…”

Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein

“…[3][4][5][6][7][8] Concomitant with the dissemination of fluorescent protein-based labeling methods, new labeling methods based on chemical biology have been proposed, such as tag-probe labeling technologies, in which synthetic fluorescent probes are attached to the target proteins carrying genetically encoded tags through specific interactions between the fluorescent probes and the tags. [9][10][11][12] These chemical biology-based approaches allow for the labeling of proteins with synthetic fluorophores bearing various properties and controlling the timing of the labeling, thus broadening their applications in protein analysis. However, these methods require labeling procedures that are specific to each technology, which hampers their wide-spread use; also, the cytotoxicity that may accompany labeling has not yet been fully understood because of their limited use.…”