Recent progress in enzymatic protein labelling techniques and their applications

Abstract

Protein-based conjugates are valuable constructs for a variety of applications. Conjugation of proteins to fluorophores is commonly used to study their cellular localization and the protein-protein interactions. Modification of therapeutic proteins with either polymers or cytotoxic moieties greatly enhances their pharmacokinetics and potency. To label a protein of interest, conventional direct chemical reaction with the side-chains of native amino acids often yields heterogeneously modified products. This renders their characterization complicated, requires difficult separation steps and may impact protein function. Although modification can also be achieved via the insertion of unnatural amino acids bearing bioorthogonal functional groups, these methods can have lower protein expression yields, limiting large scale production. As a site-specific modification method, enzymatic protein labelling is highly efficient and robust under mild reaction conditions. Significant progress has been made over the last five years in modifying proteins using enzymatic methods for numerous applications, including the creation of clinically relevant conjugates with polymers, cytotoxins or imaging agents, fluorescent or affinity probes to study complex protein interaction networks, and protein-linked materials for biosensing. This review summarizes developments in enzymatic protein labelling over the last five years for a panel of ten enzymes, including sortase A, subtiligase, microbial transglutaminase, farnesyltransferase, N-myristoyltransferase, phosphopantetheinyl transferases, tubulin tyrosin ligase, lipoic acid ligase, biotin ligase and formylglycine generating enzyme.

Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

“…Site‐specific enzymatic conjugation approaches achieve homogeneous protein modifications (16). Here, we utilized microbial transglutaminase (mTGase, E.C.…”

Site-specific C-terminal dinitrophenylation to reconstitute the antibody Fc functions for nanobodies

“…Design, synthesis and characterization of nanobody 7D12-DNP conjugates Nanobody 7D12, which recognizes human epidermal growth factor receptor (EGFR) expressed by many tumors, such as A431 cells, 38 was utilized as a model to prove our concept. The wellestablished Sortase-A (SrtA)-mediated ligation (SML) method [39][40][41][42][43][44] was used for the site-specic modication of nanobody 7D12 with DNP. SrtA is a bacterial enzyme that recognizes a specic peptide motif, LPXTG (X can be any amino acid except for cysteine), known as the sorting signal, at the protein C-terminus and cleaves the peptide bond between threonine and glycine to form a thioester intermediate, which is followed by reacting with substrates containing oligoglycine to provide the ligation product.…”

Chemical Methods for Selective Labeling of Proteins

“…Such bio‐orthogonal reactions offer a powerful tool for single‐site protein modification. The insertion of exogenous peptide or a sequence for selective recognition by a reagent an enzyme also provides an opportunity to label the genetically modified region in a protein selectively. The efforts to develop the bio‐orthogonal transformations in the last few decades have been remarkable.…”