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.

Protein lipid modification involves the attachment of hydrophobic groups to proteins via ester, thioester, amide, or thioether linkages. In this review, the specific click chemical reactions that have been employed to study protein lipid modification and their use for specific labeling applications are first described. This is followed by an introduction to the different types of protein lipid modifications that occur in biology. Next, the roles of click chemistry in elucidating specific biological features including the identification of lipid-modified proteins, studies of their regulation, and their role in diseases are presented. A description of the use of protein–lipid modifying enzymes for specific labeling applications including protein immobilization, fluorescent labeling, nanostructure assembly, and the construction of protein–drug conjugates is presented next. Concluding remarks and future directions are presented in the final section.

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