Characterization of a monoPEG20000-Endostar

In this study, we investigated the PEG attachment site of mono-PEGylated Endostar, a modified recombinant human endostatin approved in China for lung cancer. N-terminal site-directed mono-PEGylation of Endostar was accomplished using mPEG-propionaldehyde derivatives (Mw = 20 kDa) under slightly acid...

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Bibliographic Details
Published in:International journal of biological macromolecules 2010-04, Vol.46 (3), p.331-336
Main Authors: Tong, Yue, Zhong, Kai, Tian, Hong, Gao, Xiangdong, Xu, Xiangyang, Yin, Xiaojin, Yao, Wenbing
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Blotting, Western
Chromatography, High Pressure Liquid
Chromatography, Liquid
Chromatography, Reverse-Phase
Electrophoresis, Polyacrylamide Gel
Endostar
Endostatins - analysis
Endostatins - chemistry
Endostatins - isolation & purification
Humans
Location
Molecular Sequence Data
N-terminal
Peptides - chemistry
Poly(ethylene glycol) (PEG)
Polyethylene Glycols - analysis
Polyethylene Glycols - chemistry
Polyethylene Glycols - isolation & purification
Recombinant Proteins
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Trypsin - metabolism
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Summary:In this study, we investigated the PEG attachment site of mono-PEGylated Endostar, a modified recombinant human endostatin approved in China for lung cancer. N-terminal site-directed mono-PEGylation of Endostar was accomplished using mPEG-propionaldehyde derivatives (Mw = 20 kDa) under slightly acidic pH conditions (pH 5.5). One-step cation exchange chromatography was used to purify the mono-PEGylated Endostar. Following tryptic digestion, the peptide fragment containing PEG was separated by SDS-PAGE. Barium iodide staining and Western blotting were used to detect the PEG moiety and the N-terminus of Endostar, respectively. The peptide fragment stained by barium iodide showed a positive response to anti-(His) 6 mAb, demonstrating that PEG was located at the N-terminus of Endostar. LC/MS was applied to verify the occurrence of mono-PEGylation at the N-terminus of Endostar.
ISSN:0141-8130
1879-0003
DOI:10.1016/j.ijbiomac.2010.01.017