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. Author manuscript; available in PMC: 2026 Jul 18.
Published in final edited form as: Nat Chem. 2025 Jul 18;17(9):1371–1382. doi: 10.1038/s41557-025-01871-3

Figure 5. MccB enables ATP-dependent thioester formation and regeneration for high-yield enzyme-catalyzed expressed protein ligation.

Figure 5.

(A) Enzyme-catalyzed expressed protein ligation is limited by subtiligase-catalyzed hydrolysis of the thioester substrate, generating a dead-end product. We used MccB for ATP-dependent thioester formation and regeneration, enabling reactivation of the dead-end hydrolytic product. (B) High yield one-pot MccB- and subtiligasecatalyzed ATP-dependent peptide ligation to GFP-TeCH. (C) MccB- and subtiligase-catalyzed ATP-dependent peptide ligation to TeCH-tag fusions of MBP, the catalytic domain of protein tyrosine phosphatase 1B (PTP1B1–321), protein L, an α-GFP recombinant antibody, and an EGFR-targeting affibody. The * indicates an α-gluconylated form of protein L that is an artifact of His-tag purification. (D) MccB- and subtiligase-catalyzed peptide ligation and strain promoted azide-alkyne cycloaddition were used to synthesize α-GFP rAb-Cy3 for staining of a HEK293T cell line engineered for doxycycline-inducible expression of cell surface GFP. (E) Dual N- and C-terminal labeling of TeCH-tagged MBP with 5-FAM-LPETGG and AFAGAGS-azidoAla using eSrtA and MBP/subtiligase under one-pot (center) or telescoping (right) conditions.