PEGylation of native disulfide bonds in proteins
PEGylation of native disulfide bonds in proteins
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DOI：
10.1038/nprot.2006.346
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Shaunak, Sunil
中科院分区：
文献类型：
--
作者：
Brocchini, Steve;Balan, Sibu;Shaunak, Sunil
聚乙二醇化使蛋白质成为重要的新型生物药物。现有PEG化方法的基本问题是低效率的缀合和形成异质混合物。这是因为聚（乙二醇）（PEG）通常与亲核胺残基缀合。我们的PEG化方案通过利用许多生物相关蛋白质的二硫键中两个硫原子的化学反应性来解决这些问题。可接近的二硫键被温和地还原以释放两个半胱氨酸硫原子而不干扰蛋白质的三级结构。位点特异性PEG化用双硫醇烷基化PEG试剂实现，所述试剂依次经历缀合以形成三碳桥。两个硫原子与选择性缀合至桥的PEG重新连接。蛋白质的PEG化可在24小时内完成，PEG-蛋白质缀合物的纯化可在另外3小时内完成。我们已经成功地将这种方法应用于细胞因子、酶、抗体片段和肽的PEG化，而不破坏它们的三级结构或消除它们的生物活性。
PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three- carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.