Department of Biochemistry, China Pharmaceutical University, Nanjing 210009, China.
Int J Biol Macromol. 2010 Apr 1;46(3):331-6. doi: 10.1016/j.ijbiomac.2010.01.017. Epub 2010 Feb 1.
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.
在这项研究中，我们研究了单聚乙二醇化恩度的 PEG 连接点，恩度是一种经中国批准用于肺癌的改良重组人内皮抑素。在略酸性 pH 条件（pH 5.5）下，使用 mPEG-丙醛衍生物（Mw=20 kDa）对恩度进行 N 端定点单聚乙二醇化。采用一步阳离子交换层析法纯化单聚乙二醇化恩度。经胰蛋白酶消化后，通过 SDS-PAGE 分离含有 PEG 的肽段。使用碘化钡染色和 Western blot 分别检测 PEG 部分和恩度的 N 末端。碘化钡染色的肽段对抗（His）6 mAb 呈阳性反应，表明 PEG 位于恩度的 N 末端。LC/MS 用于验证恩度 N 末端单聚乙二醇化的发生。
