This study describes the biosynthesis of a human epidermal growth factor fusion protein, Long EGF, that has a 53 amino acid extension peptide derived from the 46 N-terminal amino acids of porcine GH. The approach allowed the production of Long EGF at high efficiency due to the expression of the fusion protein in high yield as inclusion bodies in Escherichia coli. Long EGF had a slightly lower potency compared with native EGF in a range of assays, including binding to anti-EGF antibodies or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat intestinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth factor-β1.
Degradation of Long EGF and native EGF was compared in gastrointestinal 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 rats caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg53 in the stomach and a removal of the C-terminal pentapeptide (49Trp-Trp-Glu-Leu-Arg53) in the small bowel was demonstrated by N-terminal sequencing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent binding activities to the EGF receptor and anti-EGF antibodies. The data show that a human EGF fusion protein can be produced efficiently in a bacterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EGF in both stomach and small bowel producing similar biological activity to native EGF, it could not prevent subsequent degradation of the EGF domain. Other strategies are being investigated to develop an effective oral form of EGF that resists digestion by proteases in the gastrointestinal tract.