Directed evolution of ATP binding proteins from a zinc finger domain by using mRNA display
Directed evolution of ATP binding proteins from a zinc finger domain by using mRNA display
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DOI：
10.1016/j.chembiol.2005.10.015
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发表时间：
2006-02-01
影响因子：
--
通讯作者：
Szostak, JW
中科院分区：
文献类型：
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作者：
Cho, GS;Szostak, JW
抗体传统上用于分离亲和试剂到新的分子靶标，但替代蛋白质支架越来越多地用于定向进化具有新的分子识别特性的蛋白质。我们设计了一个基于人维甲酸-X-受体（hRXR α）的DNA结合结构域的组合文库。我们选择这个域是因为它的尺寸小，折叠稳定，并且有两个紧密并列的识别环。我们用随机氨基酸片段替换了这两个环，并使用mRNA展示来分离特异性识别三磷酸腺苷（ATP）的变体，这表明该蛋白质结构域的功能从DNA结合到ATP识别发生了显著改变。许多新的独立序列被回收，具有中等亲和力和ATP的高特异性，验证了这种支架的功能分子的产生。
Antibodies have traditionally been used for isolating affinity reagents to new molecular targets, but alternative protein scaffolds are increasingly being used for the directed evolution of proteins with novel molecular recognition properties. We have designed a combinatorial library based on the DNA binding domain of the human retinoid-X-receptor (hRXR alpha). We chose this domain because of its small size, stable fold, and two closely juxtaposed recognition loops. We replaced the two loops with segments of random amino acids, and used mRNA display to isolate variants that specifically recognize adenosine triphosphate (ATP), demonstrating a significant alteration of the function of this protein domain from DNA binding to ATP recognition. Many novel independent sequences were recovered with moderate affinity and high specificity for ATP validating this scaffold for the generation of functional molecules.