Probing the recognition of post-translational modifications by combining sortase-mediated ligation and phage-assisted selection.
Probing the recognition of post-translational modifications by combining sortase-mediated ligation and phage-assisted selection.
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DOI：
10.1021/cb4001487
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发表时间：
2013-06
影响因子：
4
通讯作者：
T. Teschke;Bernhard Geltinger;A. Dose;C. Freund;D. Schwarzer
中科院分区：
文献类型：
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作者：
T. Teschke;Bernhard Geltinger;A. Dose;C. Freund;D. Schwarzer
可逆的翻译后修饰（PTM）是蛋白质功能的关键调节因子，并调节信号转导网络中的许多蛋白质-蛋白质相互作用。在这里，我们描述了一种策略，用于确定PTM结合蛋白的修饰偏好，只有最小的蛋白质量，可以通过免疫沉淀从哺乳动物细胞裂解物中获得。该方法基于分选酶介导的连接和噬菌体辅助选择策略的组合。该方法可用于分析介导相互作用的修饰类型以及修饰位点侧翼氨基酸的影响。我们已经证明了这种方法的适用性，通过探测磷酸化的酪氨酸和丝氨酸残基与其各自的结合域的相互作用。
Reversible post-translational modifications (PTMs) are key regulators of protein function and modulate a multitude of protein-protein interactions in signal transduction networks. Here, we describe a strategy for determining the modification preferences of PTM-binding proteins with only minimal protein amounts that can be obtained by immunoprecipitation from mammalian cell lysates. This method bases on the combination of sortase-mediated ligation and phage-assisted selection strategies. This method can be used to analyze the type of modification that mediates the interaction as well as the influence of the amino acids flanking the modification sites. We have demonstrated the applicability of this method by probing the interaction of phosphorylated tyrosine and serine residues with their respective binding domains.