Poster Presentation International Peptide Symposium 2023

Human trefoil factor 2 (hTFF2) belongs to an important family of peptides containing a well-structured trefoil domain^[1]. hTFF2 is secreted into the gastrointestinal tract where it plays important role in protecting and repairing the mucosa; it thus holds therapeutic promise for the treatment of chronic gastrointestinal disorders^[2]. hTFF2 contains 106 amino acid residues (¹⁵Asn glycosylated) and displays two trefoil domains formed by 7 disulfide bonds. Its 3D-structure, mode of action and target receptor remain unknown as only limited amounts of hTFF2 can be obtained from human tissue extraction. Here, we describe a yeast expression system designed to produce hTFF2 and its glycosylated and ¹⁵N-enriched analogues for physiological, biochemical and spectroscopic studies.

We designed the hTFF2 gene encoding a fusion protein and constructed recombinant plasmids and optimized conditions for protein expression. The secreted hTFF2 was found in a glycosylated and non-glycosylated form S. cerevisiae. We also produced a ¹⁵N-enriched analogue of hTFF2 to facilitate NMR structure determination.  Furthermore, we also describe our semi-synthesis approach to synthesize hTFF2 protein by expressed protein ligation using E. Coli based expression system. Access to large quantities and 3D structure of hTFF2 will help to elucidate its mode of action in gastrointestinal protection and wound healing.