Figure 1.

Expressed protein ligation. (A) Intein-mediated thiolysis generates a thioester group at the C terminus of the complete extracellular region of the Eph receptor. The cysteine residue at the N terminus of a synthetic peptide then reacts with the thioester group to generate the semisynthetic product. (B) Schematic representation of the fusion protein construct used for the expression. The fusion protein was generated by inserting the intein between the Eph ECD and the Fc purification tag. To increase the thiol cleavage efficiency, a Ser → Phe and two Cys → Ser mutations in the GyrA intein were created as described in the text.