Figure 1.

(A) A schematic of the intein-based protein expression and fluorescent-labeling method. EGFR-ICD and EGFR-ΔCT are each expressed as tripartite fusion proteins with intein (Int) and chitin-binding domain (CBD) sequences and immobilized on a chitin matrix. The thiol-ester intermediate is cleaved with an exogenous thiol (cysteine conjugate of the fluorophore EDANS), resulting in the release of the EGFR protein. The cysteine-conjugated EDANS is irreversibly incorporated at the extreme C terminus of EGFR-ICD/ΔCT proteins as a result of an S–N acyl transition to form a stable amide bond. (B) Structure of the cysteine-EDANS conjugate (CEDANS) used in intein-mediated labeling of the EGFR C terminus.