Figure 2

Monitoring chemical synthesis of SLURP-1 by uHPLC. (A-C) One-pot NCL of SLURP-1 segments. (A) Ligation of SLURP-1[21–50] and SLURP-1[51–81]-α-thioester segments at t = 0 and, (B) after 12 h ligation and Thz to Cys conversion. (C) SLURP-1[1–20]-α-thioester segment was subsequently added and ligated to fragment 21–81 to form the reduced SLURP-1 polypeptide. (D,E) Folding of the SLURP-1 polypeptide monitored by uHPLC. (D) Reduced SLURP-1 and (E) crude folding mixture after 70 h. The principal peak at retention time 5 min corresponds to correctly folded human SLURP-1. ESI-MS spectra of the dominant peaks are shown as inserts. (F) uHPLC and high-resolution MS analysis of purified human sSLURP-1. The inset shows an isotopically resolved blow-up of the [M + 7 H]⁷⁺ charge state.