C-terminal site-specific PEGylation of a truncated thrombomodulin mutant with retention of full bioactivity
C-terminal site-specific PEGylation of a truncated thrombomodulin mutant with retention of full bioactivity
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DOI：
10.1021/bc049903y
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发表时间：
2004-09-01
影响因子：
4.7
通讯作者：
Chaikof, EL
中科院分区：
文献类型：
--
作者：
Cazalis, CS;Haller, CA;Chaikof, EL
向生物活性蛋白中添加聚乙二醇（PEG化）可改善其血浆半衰期，增强抗蛋白水解裂解的稳定性，还可降低蛋白免疫原性。典型地，PEG化通常涉及与可用的赖氨酸氨基的反应，其中一些可以在生物活性位点内或附近。因此，大多数方案是非特异性的，并导致蛋白质活性的丧失。我们在此报告了一种策略，在C端的血栓调节蛋白（TM）衍生物的位点特异性聚乙二醇化。在大肠杆菌中表达了由表皮生长因子（EGF）样结构域4-6组成的截短的TM突变体，其C-末端具有叠氮甲硫氨酸。通过施陶丁格反应将TM突变体位点特异性缀合至甲基-PEG-三芳基膦化合物。PEG化前后TM构建体的酶活性没有变化，这证实了该位点特异性PEG化方案的实用性。
Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)like domains 4-6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme.