Rapid construction of metabolite biosensors using domain-insertion profiling.
Rapid construction of metabolite biosensors using domain-insertion profiling.
复制标题
DOI：
10.1038/ncomms12266
复制
发表时间：
2016-07-29
影响因子：
16.6
通讯作者：
Savage DF
中科院分区：
文献类型：
--
作者：
Nadler DC;Morgan SA;Flamholz A;Kortright KE;Savage DF
单荧光蛋白生物传感器（SFPBs）是一类重要的探针，能够在体内对分析物进行单细胞定量。尽管比其他检测技术具有优势，但其使用受到其构造的固有挑战的限制。具体而言，绿色荧光蛋白（GFP）插入配体结合结构域的合理设计，产生必要的变构偶联，仍然是一个限速步骤。在这里，我们描述了一种无偏见的方法，称为域插入分析与DNA测序（DIP-seq），结合快速创建不同的库的潜在的SFPBs和高通量活性测定，以确定功能的生物传感器。作为一个概念的证明，我们构建了一个SFPB的重要调节糖海藻糖。海藻糖结合蛋白的DIP-seq分析揭示了GFP插入的变构热点，并产生了在体内功能强大的高动态范围生物传感器。总之，DIP-seq同时加速了代谢物生物传感器的构建，并为询问蛋白质变构提供了一种新的工具。 在单荧光蛋白生物传感器的构建中，荧光蛋白插入配体结合结构域的插入点的选择是限速步骤。在这里，作者开发了一种无偏见的高通量方法，称为DNA测序的结构域插入分析（DIP-seq），以产生一种新型的海藻糖生物传感器。
Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery. In the construction of single fluorescent protein biosensors, selection of the insertion point of a fluorescent protein into a ligand-binding domain is a rate-limiting step. Here, the authors develop an unbiased, high-throughput approach, called domain insertion profiling with DNA sequencing (DIP-seq), to generate a novel trehalose biosensor.