Fig. 1. Semi-synthetic protein trans-splicing directed by a minimal high-affinity interaction pair. (a) Design of synthetic IN fragments with five EN residues (KKESG). TrisNTA was incorporated during Fmoc-based SPPS. 5(6)-Carboxyfluorescein (FAM) or Cy5 were used as fluorescent reporter. (b) Design of recombinant IC with thioredoxin (Trx). N-terminal His6-tagged HICT and HLICT were used for interaction analyses with IN-trisNTA and IN-L-trisNTA. ICTH with a C-terminal His6-tag served as control. (c) PTS of ICTH/HICT (10 μM) and FAM-labeled IN (40 μM) yielded fluorescently labeled Trx (1 h, 20 °C). SP, splice product; *, impurity/premature cleaved intein.20,44 .
