Synthesis of Ub-ACA, a Fluorogenic DUB Substrate via ACPL Method

This chapter details the step-by-step synthesis of ubiquitin-7-amino-4-coumarinyl-acetic acid (Ub-ACA) using the activated cysteine-directed protein ligation (ACPL) technique. Ub-ACA is a fluorogenic substrate for deubiquitinases (DUBs). It can be readily synthesized via ACPL to a high quantity by exchanging a C-terminal cysteine residue in a recombinantly expressed Ub-G76C mutant with Gly-ACA, a process mediated simply by 2-nitro-5-thiocyanatobenzoic acid (NTCB). This chapter provides protocols for protein expression, thiol activation, fluorophore conjugation, and product purification, yielding Ub-ACA with high purity suitable for enzymatic assays.