The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG) group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

Protein prenylation is a ubiquitous covalent post-translational modification found in all eukaryotic cells, comprising attachment of either a farnesyl or a geranylgeranyl isoprenoid. It is essential for the proper cellular activity of numerous proteins, including Ras family GTPases and heterotrimeric G-proteins. Inhibition of prenylation has been extensively investigated to suppress the activity of oncogenic Ras proteins to achieve antitumor activity. Here, we review the biochemistry of the prenyltransferase enzymes and numerous isoprenoid analogs synthesized to investigate various aspects of prenylation and prenyltransferases. We also give an account of the current status of prenyltransferase inhibitors as potential therapeutics against several diseases including cancers, progeria, aging, parasitic diseases, and bacterial and viral infections. Finally, we discuss recent progress in utilizing protein prenylation for site-specific protein labeling for various biotechnology applications.

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 or 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.