N-terminal site-specific mono-PEGylation of epidermal growth factor.

PURPOSE

N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group.

METHODS

The site-specific PEG conjugation was conducted ata slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an alpha-amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF.

RESULTS

The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such a cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability whe incubated with tissue homogenate.

CONCLUSION

N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.