Creation of GPCR-based chemical sensors by directed evolution in yeast
Creation of GPCR-based chemical sensors by directed evolution in yeast
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DOI：
10.1093/protein/gzi069
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发表时间：
2006-01-01
影响因子：
2.4
通讯作者：
Broach, JR
中科院分区：
文献类型：
--
作者：
Ault, AD;Broach, JR
G蛋白偶联受体（GPCR）形成一类在配体结合和灵敏度方面具有巨大多样性的生物化学传感器。为了探索配体识别的结构方面，我们进行了人UDP-葡萄糖受体（P2 Y14）在酵母菌中功能性表达的定向进化。我们试图产生新的受体亚型与配体结合的特性，这将是有用的，在实际的生物传感器的发展。整个UDP-葡萄糖受体基因的诱变产生的受体具有增加的活性，但类似的配体特异性，而随机诱变的残基在紧邻的配体结合口袋产生突变体的配体特异性改变。通过首先敏化P2 Y14受体，然后重定向配体特异性，我们能够创建适用于简单生物传感器的突变受体。我们的研究结果表明，通过定向进化策略，使用标准的酵母遗传技术改变受体配体结合特性的可行性。新型受体突变体可用于检测复杂混合物中的化学配体，并区分化学或立体化学相关的化合物。具体来说，我们展示了如何工程受体可以应用成对的方式来区分几种化学分析物，将无法区分与一个单一的受体。这些实验证明了基于嗅觉原理的检测器设计的组合方法的可行性。
G protein-coupled receptors (GPCRs) form a class of biological chemical sensors with an enormous diversity in ligand binding and sensitivity. To explore structural aspects of ligand recognition, we subjected the human UDP-glucose receptor (P2Y14) functionally expressed in the yeast Saccharomyces to directed evolution. We sought to generate new receptor subtypes with ligand-binding properties that would be useful in the development of practical biosensors. Mutagenesis of the entire UDP-glucose receptor gene yielded receptors with increased activity but similar ligand specificities, while random mutagenesis of residues in the immediate vicinity of the ligand-binding pocket yielded mutants with altered ligand specificity. By first sensitizing the P2Y14 receptor and then redirecting ligand specificity, we were able to create mutant receptors suitable for a simple biosensor. Our results demonstrate the feasibility of altering receptor ligand-binding properties via a directed evolution strategy, using standard yeast genetic techniques. The novel receptor mutants can be used to detect chemical ligands in complex mixtures and to discriminate among chemically or stereochemically related compounds. Specifically, we demonstrate how engineered receptors can be applied in a pairwise manner to differentiate among several chemical analytes that would be indistinguishable with a single receptor. These experiments demonstrate the feasibility of a combinatorial approach to detector design based on the principles of olfaction.