Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFalpha, VGF, heparin-binding EGF, and betacellulin), suggests that Leu47, which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu47 results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF1-45 core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF1-45 core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF1-45 in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF1-47 from EGF1-45 with equivalent mitogenic and receptor binding activity to EGF1-47 generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or nonphysiological amino acids (e.g. allo-Ile) for Asp46, Leu47 or Arg48. Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.
|Pages (from-to)||71 - 80|
|Number of pages||10|
|Publication status||Published - 2002|