Experimental mapping of soluble protein domains using a hierarchical approach
Experimental mapping of soluble protein domains using a hierarchical approach
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DOI：
10.1093/nar/gkr548
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发表时间：
2011-10-01
影响因子：
14.9
通讯作者：
Waldo, Geoffrey S.
中科院分区：
文献类型：
--
作者：
Pedelacq, Jean-Denis;Nguyen, Hau B.;Waldo, Geoffrey S.
探索大型多结构域蛋白质靶标的功能和3D空间通常需要复杂的实验以获得适合于结构测定的形式的靶标。存在能够从DNA文库中选择表达良好的可溶性片段的筛选方法，但需要使用自动化来最大化挑选一些好的候选物的机会。在这里，我们描述了使用的插入二氢叶酸还原酶（DHFR）载体，以选择框内片段和分裂GFP检测技术，以过滤出表达不溶性蛋白质片段的结构。通过引入IPCR步骤以产生高密度、集中的片段子文库，这种具有成本效益的方法可以在没有结构域边界的先验知识的情况下手动进行，同时允许单个氨基酸分辨率边界作图。我们将其用于磷酸肌醇-3-激酶的良好表征的p85 α亚基，以证明我们方法的稳健性和效率。然后，我们成功地将其测试到来自结核分枝杆菌的聚酮合酶PpsC上，该聚酮合酶PpsC是参与细胞包膜中复杂脂质生物合成的潜在药物靶标。已经获得了来自酰基转移酶（AT）、脱氢酶（DH）和烯酰还原酶（ER）结构域的X射线质量晶体。
Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85 alpha subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.