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 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.

Figure 1

Selected direct chemical modifications of amino acids. (A) Cysteine modification via a) disulfide exchange, b) maleimide, or c) photo-catalysed thiol-ene couplings. (B) Lysine modification via coupling with d) isothiocyanate or activation with e) sulfonyl chloride or f) fluorine-substituted aromatic esters.

Figure 2

Structures of selected UAAs bearing fluorescent, cross-linker, affinity, or bioorthogonal handles.

Scheme 1

Selected bioorthogonal reactions based on aldehyde functionality. (A) Oxime/hydrazone ligation. (B) Pictet-Spengler ligations.

Scheme 2

Selected bioorthgonal reactions based on azide functionality. (A) The Staudinger ligation. (B) The traceless Staudinger ligation. (C) CuAAC reaction. (D) SPAAC reaction. DBCO is shown as an example for the strained alkyne compound.

Scheme 3

Selected examples of tetrazine ligations. (A) Tetrazine ligation with TCO. (B) Tetrazine ligation with a strained alkyne

Scheme 4

Enzymatic labelling by SrtA. (A) Canonical C-terminal labelling catalysed by SrtA using oligoglycine substrates. (B) Labelling of the lysine (in a pilin domain) by SrtA using LPETG peptide substrates. (C) Protein labelling at the C-terminus by SrtA using primary amine or hydrazide-containing substrates. POI: protein of interest. Functionality/residues from the enzymatically added substrate are highlighted in red.

Scheme 5

Protein N-terminal labelling by Subtiligase using peptide ester substrates.

Scheme 6

Enzymatic protein labelling by MTG. (A) Labelling of target proteins containing Q-tag sequences using lysine or Primary amine Substractes. (B) Labelling of K-tag modified protein using a ZQG peptide dervatized with various cargos.

Scheme 7

Protein labelling at C-terminus of a POI terminating in a CaaX-box sequence by FTase using isoprenoid analogues bearing bioorthogonal functional groups.

Scheme 8

Protein labelling by NMT in E. coli. The plasmids expressing the POI and NMT are both transformed into E. coli. At the time of expression, myristic acid analogues are added to the culture medium, which are then converted to CoA modified substrates by endogenous enzymatic activities. Proteins are labelled in vivo at the N-terminus.

Scheme 9

PPTase-catalysed reactions for site-specific enzymatic labelling. (A) Proteins fused with PCP or ACP as recognition domains for modification of CoA derivatives. (B) Shorter tags (ybbR, S6, A1, and A4) can be inserted within exposed loops of the POI for internal modification.

Scheme 10

TTL-mediated attachment of tyrosine analogues (A) and functionalized glycines (B) to the POI engineered with a C-terminal Tub-tag.

Scheme 11

LplA-mediated protein labelling using lipoate analogues bearing various bioorthogonal functional groups to a POI containing an engineered C-terminal LplA acceptor peptide.

Scheme 12

Biotin ligase mediated-protein labelling. (A) Protein labelling using biotin ligase with biotin analogues that contain bioorthogonal functional groups. B. Biotin ligase from S. tokodaii forms a complex with its biotin-modified protein substrate bearing the BCCP domain.

Scheme 13

FGE catalysed conversion of a cysteine to an aldehyde-bearing formylglycine residue. Labelling occurs in E. coli by coexpression of the tag-fused POI and FGE. The aldehyde functional group serves as a reactive handle for Subsequent Comjugation.