Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in vitro biologic activity.
OBJECTIVE
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.


CONCLUSIONS
This study demonstrates that biologic activities of mono-PEGylated EGF isomers are highly dependent upon the site of PEGylation in EGF.
Lee
Haeshin
H
Department of Biologic Sciences Korea Advanced Institute of Science and Technology, Taejon.
Park
Tae Gwan
TG
D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000493:pharmacokinetics*; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics*; D020033:Protein Isoforms / Q000737:chemistry / Q000493:pharmacokinetics
D020033:Protein Isoforms; D011092:Polyethylene Glycols; D004815:Epidermal Growth Factor