Characterization of Human and Rat Intestinal Trefoil Factor Produced in Yeast

Abstract

Intestinal trefoil factor (ITF) from human (hITF) and rat (rITF) have been produced in Saccharomyces cerevisiae. The DNA encoding the two peptides were cloned by polymerase chain reactions (PCR) from a human normal colon library and a rat small intestinal epithelial cell library. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the rat and human ITF sequences, respectively. The leader sequence used serves to direct the fusion protein into the secretory (and processing) pathway of the cell. The secreted recombinant hITF was found in a monomer and a dimer form, whereas the rITF was only secreted as a dimer. The secreted peptides were purified by a combination of ionic exchange chromatography and preparative HPLC. From 8 L of yeast fermentation broth, 256 mg of hITF (monomer) and 133 mg of hITF (dimer) were isolated, and from 8.7 L of fermentation broth, 236 mg of rITF (dimer) was isolated. The structure of hITF (monomer), hITF (dimer), and rITF (dimer) was determined by amino acid analyses, peptide mapping, sequence analyses, and electrospray mass spectrometry analyses. In hITF (monomer) six of the seven cysteines are disulfide-linked to form 3 disulfide bridges. Mass analysis indicated that the last cysteine residue (Cys-57) did not exist as free (-SH) cysteine, but have reacted with cysteine to form an S-S linked cystine. Sequence and mass spectrometry analyses as well as peptide mapping showed that the dimer form of both hITF and rITF is mediated by a disulfide bridge between Cys-57 residues of two monomers.

Trefoil Factor 2 Requires Na/H Exchanger 2 Activity to Enhance Mouse Gastric Epithelial Repair

“…When required, the CXCR4 antagonist AMD3100 (Sigma) was added into luminal perfusate (10 M) as well as given subcutaneously (5 mg/kg in saline, 1 h prior to experiments). In some experiments, the rat trefoil factor 3 peptide (rTFF3, 2 mg/ml in saline, solubilized on the day of the experiment) was topically applied to the exposed gastric surface at two to three times within 20 min (29). We are very grateful to Dr. Daniel Podolsky for supplying the rTFF3 peptide.…”

Intestinal Trefoil Factor Induces Decay-Accelerating Factor Expression and Enhances the Protective Activities Against Complement Activation in Intestinal Epithelial Cells

“…In some experiments, T84 cells were grown on inserts of 0.4 mm pore size (Falcon 3090; BD Biosciences, Mountain View, CA), and used after achieving transepithelial resistance Ͼ400⍀ determined with a Millicell-ERS (Millipore, Bedford, MA) apparatus. Purified recombinant human ITF was produced as described previously (28).…”

Extraction and purification of biologically active intestinal trefoil factor from human meconium

“…4 The distinct threelooped secondary structure contributes to the remarkable resistance of trefoil factors to acid and proteolytic digestion, enables them to function in the harsh environment of the gastrointestinal tract lumen, and allows them to remain biologically active after enteral administration. 5,6 We have previously demonstrated that significant amounts of TFF3/ITF are present in the meconium of human infants at different gestational ages. 7 However, whether secreted TFF3/ITF in the human meconium is biologically active remains to be tested.…”