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Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in vitro biologic activity
1Department of Biologic Sciences Korea Advanced Institute of Science and Technology, Taejon.
Purpose:
To isolate mono-PEGylated epidermal growth factor (EGF) isoforms, identify the site of PEGylation, and evaluate the biologic activity of each isoform.
Methods:
EGF was PEGylated with an NHS-PEG derivative (Mw 3,400). Mono-PEGylated EGF fraction was separated by gel-filtration HPLC and three mono-PEGylated EGF isoforms were purified by RP-HPLC. Tryptic digestion mapping of both EGF and mono-PEGylated EGF isoforms was performed to identify the PEGylation sites using RP-HPLC. The digested fragments were also analyzed by matrix-assisted laser desorption and ionization time of flight (MALDI-TOF) mass spectroscopy for further verification of the three PEG conjugation sites. The biologic activity of positional isoforms was evaluated by a cell proliferation assay and a receptor tyrosine kinase activity assay to determine the effect of PEGylation site on its activity.
Results:
Mono-PEGylated EGF was composed of three positional isomers. Tryptic digestion mapping and MALDI-TOF analysis permitted the identification of the PEGylated site of the three isoforms at N-terminus. Lysine 28, and Lysine 48. PEG-N-terminus EGF, among the three positional isomers, showed the highest activity in a cell proliferation assay and in a receptor-binding assay.
Conclusion:
This study demonstrates that biologic activities of mono-PEGylated EGF isomers are highly dependent upon the site of PEGylation in EGF.
