Chemoenzymatic Bio‐orthogonal Chemistry for Site‐Specific Double Modification of Recombinant Thrombomodulin

Abstract

Best of both worlds: A one‐pot strategy for site‐specific PEGylation through strain‐promoted alkyne–azide cycloaddition (SPAAC) and fluorescent labeling through sortase A‐mediated ligation (SML) of recombinant thrombomodulin without prior chemical modification and without diminishing the protein activity has been developed.

Flow‐Based Enzymatic Ligation by Sortase A

“…Sortagging methodology plays a vital role in the synthesis of complex bioconjugates with important structural and biological properties. Lipids,5 fluorophores,6 affinity tags,6 reactive handles,7 sugars and GPI anchors,8 polymer initiators,9 peptide nucleic acids,10 and thioesters11 have all been successfully conjugated. Methods such as surface modification12 and protein immobilization,13 protein14 and peptide15 cyclization, NN and CC protein fusion synthesis,16 chemoenzymatic protein synthesis,17 CRECS strategies (combined recombinant, enzymatic, and chemical synthesis),18 combined protein purification and modification,19 site‐specific antibody labeling,20 functionalization of PEGylated capsules,21 intracellular22 and cell‐surface labeling,23 protein‐dendrimer formation,24 sortase‐mediated hydrazinolysis,25 and production of receptor‐directed chimeras,26 among others,27 have all utilized sortase A‐mediated ligation.…”

Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy

“… Recombinant human thrombomodulin that exhibits an azidohomoalanine residue and the SrtA recognition sequence (TM 456 AL) was dually labeled by combining the copper‐free 1,3‐dipolar cycloaddition using azadibenzocyclooctyne (DIBAC)–AlexaFluor 647 and the sortase‐mediated transpeptidation by applying a BODIPY‐labeled diglycine. Success of the specific labeling was proven by SDS‐PAGE 47…”

“…Sortase A-mediated transpeptidation is readily used to label proteins with peptides, fluorophores, biotin, radioactive metal complexes, polyethyleneglycol derivatives, multimeric protein-tags, like streptavidin and peptide thioesters that can be subsequently used in NCL ( Figure 3). [39][40][41][42][43][44][45][46][47][48] Even solubility enhancement tags like GB1 can be ligated to, for example, isotopically labeled target proteins. The advantage of the latter technique over current biomolecular methods is the elimination of NMR signals arising from the tag.…”

“…Success of the specific labeling was proven by SDS-PAGE. [47] glycine-containing glycopeptides pure sugars appended with a 6-aminohexose moiety are also applicable as nucleophilic substrates. Even oligosaccharides that include a 6-deoxy-6-aminohexose moiety can be ligated to synthetic peptides or expressed proteins that exhibit the SrtA recognition sequence.…”

Flow‐Based Enzymatic Ligation by Sortase A

“…Sortagging methodology plays a vital role in the synthesis of complex bioconjugates with important structural and biological properties. Lipids, [5] fluorophores, [6] affinity tags, [6] reactive handles, [7] sugars and GPI anchors, [8] polymer initiators, [9] peptide nucleic acids, [10] and thioesters [11] have all been successfully conjugated. Methods such as surface modification [12] and protein immobilization, [13] protein [14] and peptide [15] cyclization, N À N and C À C protein fusion synthesis, [16] chemoenzymatic protein synthesis, [17] CRECS strategies (combined recombinant, enzymatic, and chemical synthesis), [18] combined protein purification and modification, [19] site-specific antibody labeling, [20] functionalization of PEGylated capsules, [21] intracellular [22] and cell-surface labeling, [23] protein-dendrimer formation, [24] sortase-mediated hydrazinolysis, [25] and production of receptor-directed chimeras, [26] among others, [27] have all utilized sortase A-mediated ligation.…”