Preparation and characterization of mono-PEGylated epidermal growth factor: Evaluation of in vitro biologic activity

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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.
Publisher
KLUWER ACADEMIC/PLENUM PUBL
Issue Date
2002-06
Language
English
Article Type
Article
Citation

PHARMACEUTICAL RESEARCH, v.19, no.6, pp.845 - 851

ISSN
0724-8741
DOI
10.1023/A:1016113117851
URI
http://hdl.handle.net/10203/13173
Appears in Collection
BS-Journal Papers(저널논문)
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