Fig. 23.

Chemoenzymatic labeling strategies of the protein of interest (POI) using post-translational modification enzymes. a Formylglycine generating enzyme (FGE) recognizes LCXPXR peptide motif and converts the side chain of Cys residue into an aldehyde group. The POI fused to the aldehyde tag can be further functionalized with aminooxy or hydrazide probes. b Farnesyltransferase (FTase) recognizes the four AAs sequence CA₁A₂X (A₁ and A₂ are non-charged aliphatic AAs and X is C-terminal Met, Ser or Phe) at the C-terminus and catalyzes the attachment of the farnesyl isoprenoid group to the Cys residue. The POI can be further labeled by bioorthogonal chemical conjugation of the farnesyl moiety functionalized with azide or alkyne. c N-Myristoyl transferase (NMT) recognizes the GXXXS peptide motif at the N-terminus and attaches a myristate group to an N-terminal Gly residue. The POI can be further labeled by bioorthogonal chemical conjugation of myristate moiety functionalized with azide or alkyne. d Biotin ligase recognizes the GGLNDIFEAQKIEWH peptide motif derived from biotin carboxyl carrier protein and catalyzes the transfer of biotin from an ATP intermediate (biotinyl 5′-adenylate) to Lys residue. Biotinylated POI can then be labeled with streptavidin conjugated with a variety of chemical probes. e Lipoic acid ligase recognizes the GFEIDKVWYDLDA peptide motif and catalyzes the attachment of lipoic acid or its derivatives to Lys residue in the motif. f Transglutaminase (TGase) catalyzes the transamination reaction and forms an iso-peptide bond between Gln in POI and Lys residue-functionalized small molecule probes, peptides or proteins. g Sortase cleaves LPXTG peptide tag fused to POI between Thr and Gly residue and conjugates oligo Gly-functionalized small molecule probes, peptides or proteins to POI by forming a peptide bond between Thr and Gly residues. h GST catalyzes Cys arylation and conjugates probes bearing a 4-mercaptoperfluorobiphenyl moiety to the N-terminal γ-Glu-Cys-Gly sequence of POI. i SpyLigase catalyzes the generation of an isopeptide bond between Lys residue in KTag and Asp residue in SpyTag