Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex*
Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex*
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DOI：
10.1074/mcp.o114.044412
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发表时间：
2014-11
影响因子：
7
通讯作者：
D. Sui;Xinjing Xu;Xuemei Ye;Mengyu Liu;Maxwell Mianecki;Chotirat Rattanasinchai;C. Buehl;Xiexiong Deng;M. Kuo
中科院分区：
文献类型：
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作者：
D. Sui;Xinjing Xu;Xuemei Ye;Mengyu Liu;Maxwell Mianecki;Chotirat Rattanasinchai;C. Buehl;Xiexiong Deng;M. Kuo
由于在大肠杆菌中生产困难，许多生物医学关键蛋白质在蛋白质组学和生化研究中代表性不足。这些蛋白质可能具有对其功能至关重要的翻译后修饰，往往会错误折叠并被分割成细菌包涵体，或者仅在化学计量二聚体复合物中发挥作用。这些蛋白质的成功生产需要这些蛋白质与特定的“促进剂”之间的有效相互作用，例如蛋白质修饰酶、分子伴侣或二聚体复合物内的天然物理伙伴。在这里，我们报告了蛋白质相互作用模块辅助功能X（PIMAX）系统的设计和应用，该系统有效地克服了这些障碍。通过将两种感兴趣的蛋白质融合到一对经过充分研究的蛋白质-蛋白质相互作用模块中，我们能够增强这两种蛋白质的关联，从而成功产生具有酶活性的细胞周期蛋白依赖性激酶复合物和过度磷酸化的 tau 蛋白，这与阿尔茨海默病密切相关。此外，通过 PIMAX 使用 GSK-3β 和 CDK5 激酶定量磷酸化的 tau 亚型，我们在体外证明了过度磷酸化刺激的 tau 寡聚化，为新的阿尔茨海默病药物发现铺平了道路。 PIMAX 的矢量可以轻松修改以满足不同应用的需求。因此，这种方法提供了一个方便的模块化套件，对蛋白质组学和生物医学研究具有广泛的影响。
Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific “facilitator,” such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module–assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein–protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.