Fig. 3. (A) Scheme for the second ligation and folding to afford the SelF(Q74A) (8). Reaction conditions: 2^nd NCL, 6.0 M Gdn·HCl, 0.2 M Na₂HPO₄, 5 mM TCEP, 0.1 M sodium ascorbate, 50 mM MPAA, pH 6.5, RT, 12 h; refolding reaction, 0.4 M Arg·HCl, 0.2 M Tris, 0.1 M (NH₄)₂SO₄, 2 mM EDTA, 0.2 mM GSSG, 1 mM GSH, pH 8.2, 4 °C, 12 h. (B) (a and b) Analytical HPLC traces of ligation at 1 min and 12 h, respectively. * denotes the thiolactone derivative of peptide 1. Inset a: SDS-PAGE analysis of the ligation reaction. Inset b: ESI-MS of 7. (c) Analytical HPLC trace of the refolding at 12 h and ESI-MS of 8. # denotes a mixture of hydrolyzed 1, unreacted peptide 6 and the misfolded by-products, etc. + denotes a small amount of deselenized by-product. (d) Analytical HPLC trace of the purified 8. Inset: CD spectra of the synthetic SelF(Q74A) and the expressed SelF(U65C) (left), SDS-PAGE (right) of 8.