MccB‐catalyzed C‐terminal Thioesterification for Protein Bioconjugation

Protein bioconjugation enables the installation of functional groups, probes, and payloads that cannot be genetically encoded, thereby enabling the study and manipulation of biological systems. An important class of intermediates in protein bioconjugation is C-terminal protein α-thioesters, which are kinetically stable yet thermodynamically activated as electrophiles for C-terminal functionalization. An ATP-driven, MccB-catalyzed enzymatic system for synthesis of protein C-terminal thioesters was recently developed to complement existing tools for chemical and intein-mediated synthesis of C-terminal thioesters. This system relies on modification of the target protein with a thioesterification C-terminal handle (TeCH tag) that is specifically recognized by MccB, enabling conversion of the C-terminal α-carboxylate to an α-thioester. Thioesters generated with the MccB/TeCH tag system can be integrated with well-established bioconjugation methods, including expressed protein ligation and enzyme-catalyzed expressed protein ligation, expanding the scope and versatility of this approach. This article outlines the steps for applying the MccB/TeCH tag system for epitope-specific C-terminal thioesterification and for the use of MccB-generated thioesters for expressed protein ligation and enzyme-catalyzed expressed protein ligation. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: MccB-catalyzed protein C-terminal thioesterification Alternate Protocol 1: MccB-catalyzed expressed protein ligation Alternate Protocol 2: Stepwise MccB-catalyzed protein C-terminal thioesterification followed by expressed protein ligation Support Protocol 1: Expression and purification of MccB Basic Protocol 2: MccB- and subtiligase-catalyzed expressed protein ligation.