非变性聚丙烯酰胺凝胶电泳（Native PAGE）消除了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中聚乙二醇（PEG）与十二烷基硫酸钠（SDS）相互作用的问题，并为蛋白质聚乙二醇化的表征提供了一种替代高效液相色谱（HPLC）的方法。
Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation.
作者信息
Zheng Chunyang, Ma Guanghui, Su Zhiguo
机构信息
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, PR China.
出版信息
Electrophoresis. 2007 Aug;28(16):2801-7. doi: 10.1002/elps.200600807.
PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.
近年来，蛋白质聚乙二醇化已成为一项日益重要的技术。然而，聚乙二醇化产物的测定和表征存在问题，尤其是对于含有各种修饰蛋白、未反应的聚乙二醇和未修饰蛋白的反应混合物。采用两种高效液相色谱法和两种电泳法对人血清白蛋白（HSA）与分子量为5000、10000和20000的聚乙二醇进行聚乙二醇化反应混合物的表征进行了比较研究。反相高效液相色谱法（RP-HPLC）无法提供有关分子量为20000的聚乙二醇反应的正确信息。尺寸排阻高效液相色谱法（SE-HPLC）对分子量为5000的聚乙二醇反应的分离效果很差。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）可以对所有聚乙二醇化的多个样品进行分析，但条带可能由于聚乙二醇与十二烷基硫酸钠之间的相互作用而出现拖尾或变宽。另一方面，非变性聚丙烯酰胺凝胶电泳（native PAGE）消除了聚乙二醇-十二烷基硫酸钠相互作用的问题，并为所有样品提供了更好的分离效果。在非变性条件下，各种聚乙二醇化产物和未修饰的蛋白在非变性聚丙烯酰胺凝胶电泳中迁移情况不同。结果表明，对于聚乙二醇化蛋白缀合物的分析，尤其是对于聚乙二醇化混合物的表征，非变性聚丙烯酰胺凝胶电泳可以成为高效液相色谱法和十二烷基硫酸钠聚丙烯酰胺凝胶电泳的良好替代方法。
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