Rapid creation of a novel protein function by in vitro coevolution
Rapid creation of a novel protein function by in vitro coevolution
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DOI：
10.1016/j.jmb.2005.02.070
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发表时间：
2005-05-20
影响因子：
5.6
通讯作者：
Zhao, HM
中科院分区：
文献类型：
--
作者：
Chen, ZL;Zhao, HM
我们开发了一种简单有效的方法，可以在现有的蛋白质支架中创建新的蛋白质功能。体外协同进化方法涉及为目标功能设计一条假设途径，然后沿着该途径逐步定向进化相应的蛋白质。作为测试案例，该策略用于设计具有新型皮质酮活性的人雌激素受体α配体结合域（hERαLBD）的变体。选择两种类固醇，睾酮和黄体酮，在 17 β-雌二醇和皮质酮之间提供渐进的结构桥梁，以协助 hER α LBD 的定向进化。在四轮随机诱变中总共筛选了大约 106 个变体，产生了两个对皮质酮有反应的 hERaLBD 变体。这种新配体活性的产生需要四个同时突变的存在。此外，几个必需的突变位于配体结合袋之外，但对配体结合发挥了重要作用。我们的结果证明了体外共同进化创造新蛋白质功能的能力，这是现有蛋白质工程方法难以或不可能实现的，并且还揭示了核激素受体的自然进化。这种体外共同进化方法应该为工程具有新功能的生物分子和系统提供强大的、广泛适用的工具。 (c) 2005 Elsevier Ltd. 保留所有权利。
We have developed a simple and efficient method for creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves design of a hypothetical pathway for the target function followed by stepwise directed evolution of the corresponding protein along the pathway. As a test case, this strategy was used to engineer variants of human estrogen receptor alpha ligand-binding domain (hER alpha LBD) with novel corticosterone activity. Two steroids, testosterone and progesterone, that provide a progressive structural bridge between 17 beta-estradiol and corticosterone, were chosen to assist the directed evolution of hER alpha LBD. A total of approximately 106 variants were screened in four rounds of random mutagenesis, resulting in two hERaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches and also shed light on the natural evolution of nuclear hormone receptors. This in vitro coevolution approach should provide a powerful, broadly applicable tool for engineering biological molecules and systems with novel functions. (c) 2005 Elsevier Ltd. All rights reserved.