PRODUCTION OF A HUMAN EPIDERMAL GROWTH-FACTOR FUSION PROTEIN AND ITS DEGRADATION IN RAT GASTROINTESTINAL FLUSHINGS

This study describes the biosynthesis of a human epidermal growth fact or fusion protein, Long EGF, that has a 53 amino acid extension peptid e derived from the 46 N-terminal amino acids of porcine GH. The approa ch allowed the production of Long EGF at high efficiency due to the ex pression of the fusion protein in high yield as inclusion bodies in Es cherichia coli. Long EGF had a slightly lower potency compared with na tive EGF in a range of assays, including binding to anti-EGF antibodie s or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat inte stinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth fac tor-beta 1. Degradation of Long EGF and native EGF was compared in gas trointestinal flushings as an indication of whether the EGF domain of the fusion protein would be protected from proteolytic cleavage and be useful as a trophic agent in the gut. Incubation with flushings from the stomach or jejunum of raps caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg(53) in t he stomach and a removal of the C-terminal pentapeptide ((49)Trp-Trp-G lu-Leu-Arg(53)) in the small bowel was demonstrated by N-terminal sequ encing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent bind ing activities to the EGF receptor and anti-EGF antibodies. The data s how that a human EGF fusion protein can be produced efficiently in a b acterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EG F in both stomach and small bowel producing similar biological activit y to native EGF, it could not prevent subsequent degradation of the EG F domain. Other strategies are being investigated to develop an effect ive oral form of EGF that resists digestion by proteases in the gastro intestinal tract.