SARCOSYL-PAGE: a new electrophoretic method for the separation and immunological detection of PEGylated proteins.
SARCOSYL-PAGE: a new electrophoretic method for the separation and immunological detection of PEGylated proteins.
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DOI：
10.1007/978-1-61779-821-4_7
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发表时间：
2012-01-01
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影响因子：
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通讯作者：
Reichel, Christian
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作者：
Reichel, Christian
重组蛋白和合成肽的聚乙二醇化旨在产生具有改变的物理性质的生物药物。所述修饰可导致由受体介导的内吞作用减少引起的血清半衰期延长和/或由药物的流体动力学体积增加引起的肾清除延迟。MIRCERA是一种聚乙二醇化重组促红细胞生成素（rhEpo），经常用于治疗慢性肾病引起的贫血，也被运动员滥用为提高成绩的药物。虽然它可以通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹检测，但与其他促红细胞生成素相比，该检测的灵敏度显着降低。通过在样品和运行缓冲液中用肌氨酸取代SDS，SDS与蛋白质PEG基团之间的相互作用不再降低单克隆抗Epo抗体（克隆AE 7A 5）与蛋白质链的亲和力。与SDS相反，肌氨酰仅与PEG化蛋白的氨基酸链结合，从而导致增强的抗体结合和更尖锐的电泳带。虽然该方法最初是为了反兴奋剂目的而开发的，但它也可用于其他聚乙二醇化蛋白质及其电泳分离和免疫检测。
PEGylation of recombinant proteins and synthetic peptides aims to generate biopharmaceuticals with altered physical properties. The modification may lead to a prolonged serum half-life caused by a decreased receptor-mediated endocytosis and/or a delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEpo) frequently used in the treatment of anemia due to chronic kidney disease, has been also abused by athletes as a performance-enhancing drug. While it can be detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the sensitivity of the test is significantly lower compared to other epoetins. By replacing SDS with sarcosyl in the sample and running buffers, the interaction between SDS and the PEG group of the protein no longer reduces the affinity of the monoclonal anti-Epo antibody (clone AE7A5) to the protein chain. Contrary to SDS, sarcosyl only binds to the amino acid chain of the PEGylated protein, thus leading to enhanced antibody binding and a sharper electrophoretic band. While the method was originally developed for anti-doping purposes, it may be also useful for other PEGylated proteins and their electrophoretic separation and immunological detection.