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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 3. Fusion of the Thioesterification C-terminal Handle (TeCH-tag) to proteins enables MccB-catalyzed, ATP-dependent formation of C-terminal thioesters.

Figure 3.

(A) Fusion of the TeCH-tag to GFP for C-terminal thioesterification. (B) MccB catalyzes ATP-dependent thioesterification of GFP-TeCH-tag within 30 min. (C) MccB catalyzes C-terminal thioesterification of 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 catalyzes C-terminal thioesterification of GFP-TeCH-tag with cysteine, with subsequent S-to-N acyl shift leading to formation of a peptide bond as evidenced by the maleimide reactivity of the bioconjugate. (E) GFP-TeCH-tag can be modified by expressed protein ligation via a Mesna thioester intermediate in a one-pot reaction with MccB, ATP, Mesna, and the peptide CGAGS-azidoalanine.