N-terminal site-specific mono-PEGylation of epidermal growth factor
N-terminal site-specific mono-PEGylation of epidermal growth factor
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DOI：
10.1023/a:1023402123119
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发表时间：
2003-05-01
影响因子：
3.7
通讯作者：
Park, TG
中科院分区：
文献类型：
--
作者：
Lee, H;Jang, IH;Park, TG
目的.使用聚乙二醇（PEG）衍生物（Mw = 2000和5000）通过反应性末端醛基实现重组人表皮生长因子（EGF）的N-末端位点特异性单PEG化。位点特异性PEG缀合在微酸性pH条件（pH 5.5）下进行。单PEG化靶向EGF N末端的α-胺基，以最大限度地减少生物活性的降低。采用胰蛋白酶消化图谱和MALDI-TOF MS技术检测EGF N端单聚乙二醇化的发生。与天然EGF相比，位点特异性单PEG化EGF完全保留了其体外生物活性，如细胞增殖和细胞内信号转导。这表明，尽管PEG的合成聚合物共价缀合至EGF，但细胞内PEG化EGF受体的内化复合物并不妨碍细胞内信号转导事件。PEG修饰的EGF在体内也表现出延长的血流循环，并且与组织匀浆孵育时显著增强了物理稳定性。N-末端单聚乙二醇化的EGF显示出增加的物理稳定性，同时保留其生物活性。
Purpose. N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol ( PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group.Methods. The site-specific PEG conjugation was conducted at a slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an alpha-amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF.Results. The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such as cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability when incubated with tissue homogenate.Conclusion. N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.