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 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.
Thim
L
L
Novo Nordisk A/S, Bagsvaerd, Denmark.
Wöldike
H F
HF
Nielsen
P F
PF
Christensen
M
M
Lynch-Devaney
K
K
Podolsky
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006133:Growth Substances / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007422:Intestines / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3
D006133:Growth Substances; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides; D011994:Recombinant Proteins; C099037:TFF3 protein, rat; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3