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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches
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Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches

机译：用于创建基因开关的特定受体-配体对的定向进化

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摘要

Despite their versatility and power in controlling gene regulation in nature, nuclear hormone receptors (NHRs) have largely eluded utility in heterologous gene regulation applications such as gene therapy and metabolic engineering. The main reason for this void is the pleiotropic interference of the receptor-ligand combination with regulatory networks in the host organism. In recent years, numerous strategies have been developed to engineer ligand-receptor pairs that do not cross-interact with host regulatory pathways. However, these strategies have either met with limited success or cannot be readily extended to other ligand-receptor pairs. Here, we present a simple, effective, and readily generaliz-able strategy for reengineering NHRs to respond specifically to a selected synthetic ligand. The method involves generation of genetic diversity by stepwise individual site saturation mutagen-esis of a fixed set of ligand-contacting residues and random point mutagenesis, followed by phenotypic screening based on a yeast two-hybrid system. As a test case, this method was used to alter the specificity of the NHR human estrogen receptor a in favor of the synthetic ligand 4,4′-dihydroxybenzil, relative to the natural ligand 17β-estradiol, by >10~7-fold. The resulting ligand-receptor pair is highly sensitive to the synthetic ligand in human endome-trial cancer cells and is essentially fully orthogonal to the wild-type receptor-natural ligand pair. This method should provide a powerful, broadly applicable tool for engineering receptors/enzymes with improved or novel ligand/substrate specificity.
机译：尽管核激素受体（NHR）在自然界中具有控制基因调控的多功能性和力量，但在诸如基因治疗和代谢工程等异源基因调控应用中却大受追捧。该空隙的主要原因是受体-配体组合与宿主生物体中的调节网络的多效性干扰。近年来，已开发出许多策略来工程化不与宿主调控途径交叉相互作用的配体-受体对。然而，这些策略要么取得了有限的成功，要么不能轻易扩展到其他配体-受体对。在这里，我们提出了一种简单，有效且易于推广的策略，用于重新构建NHR，以对选定的合成配体作出特异性反应。该方法包括通过固定的一组配体接触残基的逐步单个位点饱和诱变和随机点诱变来产生遗传多样性，然后基于酵母双杂交系统进行表型筛选。作为测试用例，该方法用于相对于天然配体17β-雌二醇将NHR人雌激素受体a的特异性改变为有利于合成配体4,4'-二羟基苯甲醚> 10〜7倍。所得的配体-受体对对人子宫内膜癌细胞中的合成配体高度敏感，并且基本上与野生型受体-天然配体对完全正交。该方法应为工程改造具有改进的或新颖的配体/底物特异性的受体/酶提供强大而广泛适用的工具。

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