Enzymatic removal of N-glycans by PNGase F coated magnetic microparticles
Enzymatic removal of N-glycans by PNGase F coated magnetic microparticles
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DOI：
10.1002/elps.201500575
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发表时间：
2016-05-01
期刊：
影响因子：
2.9
通讯作者：
Guttman, Andras
中科院分区：
文献类型：
--
作者：
Bodnar, Judit;Szekrenyes, Akos;Guttman, Andras
蛋白质糖基化的研究是生物标志物发现和生物制药研究的重要领域。蛋白质 N-糖基化的改变可以表明医学领域病理状况或生物治疗药物生产参数的变化。这些学科的快速发展需要快速、高通量和可重复的方法来分析蛋白质 N-糖基化。目前使用的方法需要较长的去糖基化时间或大量过量的酶。在本文中，我们报告了将 PNGase F 固定到磁性微粒表面的用途及其在快速有效地从糖蛋白中去除 N-聚糖的用途。固定化 PNGase F 的使用还允许酶包被的珠子重复使用，因为在每次去糖基化反应后，磁性微粒可以通过磁铁轻松地从样品中分离出来。将 PNGase F 包被的磁珠的效率和活性与使用具有主要 N-聚糖类型（中性、高甘露糖和高度唾液酸化碳水化合物）的标准糖蛋白的溶液内酶反应进行比较。 PNGase F 包被的磁珠在 10 分钟反应时间内为免疫球蛋白 G（主要是中性碳水化合物）、核糖核酸酶 B（高甘露糖型糖）和胎球蛋白（高度唾液酸化寡糖）的模型糖蛋白提供了与传统溶液内方法相当的去糖基水平，具有易于从反应混合物中去除酶和可重复使用的特殊功能。
Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.