Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions
Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions
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DOI：
10.1074/mcp.m500309-mcp200
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发表时间：
2006-03-01
影响因子：
7
通讯作者：
Liu, B
中科院分区：
文献类型：
--
作者：
Bidlingmaier, S;Liu, B
尽管翻译后修饰（例如磷酸化）介导细胞内的基本生物学过程，但存在相对较少的方法，这些方法允许全蛋白质组鉴定与这些修饰相互作用的蛋白质。我们构建了一个酵母表面播放的人cDNA文库，并利用它来鉴定具有源自表皮生长因子受体或局灶性粘附激酶的主要酪氨酸自磷酸化位点的磷酸化肽的蛋白质片段。我们从适配器蛋白AP，磷酸肌醇3-激酶调节亚基3，SH2B和Tensin中鉴定出编码SRC同源性2个结构域的CDNA，证明了这种方法的有效性。我们的结果表明，功能性人蛋白片段的大量文库可以有效地显示在酵母表面上。除了对翻译后修饰的分析外，酵母表面脱落的人cDNA文库还具有许多潜在的应用，包括识别靶标和定义小分子或药物的潜在交叉反应蛋白。
Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs.