One-Pot Dual Labeling of IgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination of Sortase A and Butelase 1

Abstract

Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule.

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

“…Transpeptidases are appealing tools for bioconjugation, 5 , 6 , 13 , 14 , 16 , 21 , 26 , 39 , 42 but their reactions are reversible, and thus a large ratio of labeling agents to protein substrate is needed to achieve high conversion. 21 , 23 , 32 , 53 , 54 , 60 , 69 While the use of backbone-modified labels such as (thio)depsipeptides has improved yields of reactions catalyzed by sortase, 33 , 34 subtiligase, 70 , 71 trypsiligase 22 , 25 and AEPs, 32 stability of these alternative labels varies significantly and can be difficult to prepare. 50 Furthermore, the backbone-modified approach is largely limited to N-terminal labeling.…”

“…The development in this technology complements the use of existing transpeptidases such as sortase, as the differences in substrate specificity may be fully exploited in combination to develop orthogonal ligation strategies. 23 , 24 , 44 In summary, the pairing of enzymatic transpeptidation with well-established chemical reactions offers a versatile and efficient approach to the preparation of tailored protein constructs.…”

“…Protein-based approaches offer the ability to function efficiently under mild reaction conditions. 5,6 Transferase, [13][14][15] oxidoreductase, [16][17][18] ligase, 19,20 transpeptidase 2,4,[21][22][23][24][25][26][27] and (split-)intein [28][29][30] have been applied for protein bioconjugation. Nevertheless, adapting these enzymes or proteins for synthetic applications, which deviate from their natural function, oen results in technical issues.…”

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

“…MCoAEP2 can cyclize an engineered MCoTI-II scaffold. AEPs (like butelase 1 and OaAEP1 b ) have previously been shown to have a wide range of biotechnological applications, including peptide/protein ligation, peptide macrocyclization, and cell surface labeling [32][33][34][35][36][37] ; however, their utility in grafted peptide production is unclear 38 . As MCoAEP2 can produce native MCoTI-II in vitro, we hypothesized this enzyme could function as an ideal cyclase for grafted peptide production.…”

Advances in Site-specific and Linkage-specific Ligation

“…It belongs to the asparaginyl endoprotease (AEP) family, but it is virtually devoid of protease activity. Butelase 1 recognizes a C-terminal Asx-containing tripeptide motif, Asn/Asp-HisVal, to form an Asx-Xaa peptide bond where Xaa is any amino acid, either intramolecularly or intermolecularly, yielding, respectively, head-to-tail cyclic peptides or site-specific modified proteins [5][6][7][8][9][10][11][12][13][14]. Figure 2).…”