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 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.
Tong
Yue
Y
Department of Biochemistry, China Pharmaceutical University, Nanjing 210009, China.
Zhong
Kai
K
Tian
Hong
H
Gao
Xiangdong
X
Xu
Xiangyang
X
Yin
Xiaojin
X
Yao
Wenbing
W
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D043169:Endostatins; D010455:Peptides; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D014357:Trypsin; C522911:endostar protein; C549812:mono-PEGylated Endostar