CHARACTERIZATION OF HUMAN AND RAT INTESTINAL TREFOIL FACTOR PRODUCED IN YEAST

Intestinal trefoil factor (ITF) from human (hITF) and rat (rITF) have been produced in Saccharomyces cerevisiae. The DNA encoding the two pe ptides were cloned by polymerase chain reactions (PCR) from a human no rmal colon library and a rat small intestinal epithelial cell library. Recombinant plasmids were constructed to encode a fusion protein cons isting of a hybrid leader sequence and the rat and human ITF sequences , respectively. The leader sequence used serves to direct the fusion p rotein into the secretory (and processing) pathway of the cell. The se creted recombinant hITF was found in a monomer and a dimer form, where as the rITF was only secreted as a dimer. The secreted peptides were p urified by a combination of ionic exchange chromatography and preparat ive HPLC. From 8 L of yeast fermentation broth, 256 mg of hITF (monome r) and 133 mg of hITF (dimer) were isolated, and from 8.7 L of ferment ation broth, 236 mg of rITF (dimer) was isolated. The structure of hIT F (monomer), hITF (dimer), and rITF (dimer) was determined by amino ac id analyses, peptide mapping. sequence analyses, and electrospray mass spectrometry analyses. In hITF (monomer) six of the seven cysteines a re disulfide-linked to form 3 disulfide bridges. Mass analysis indicat ed 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 cystin e. Sequence and mass spectrometry analyses as well as peptide mapping showed that the dimer form of both hITF and rITF is mediated by a disu lfide bridge between Cys-57 residues of two monomers.