Protein sensor design for the quantification of heme concentration in cells

用于定量细胞中血红素浓度的蛋白质传感器设计

• 批准号: 2268795
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2268795
• 关键词: Protein; sensor; design; quantification; heme

Due to the potentially toxic nature and hydrophobicity of the heme metalloporphyrin, the necessity for this prosthetic group to be trafficked across cell membranes suggests the existence of highly controlled delivery mechanisms. Furthermore, accumulating evidence on the signaling and regulatory properties of heme is indicative of a much wider spectrum of roles for this ubiquitous cofactor than traditionally thought. New insights into the biological significance of the labile heme pool are required in order to elucidate heme roles within the cell metabolism and homeostasis. As a contribution to this grand scheme, the aim of the project is to design and build a sensor for the quantification of labile heme concentration in cells. This will involve the construction of fluorescently-labelled proteins which can bind heme under a range of heme concentrations and conditions. Moreover, the optical properties of the fusion heme-binding proteins will be exploited for imaging purposes, trying to track heme into cells.Recombinant proteins will be mainly expressed in E. Coli making use of properly engineered plasmid vectors. Purification and characterization will involve standard molecular biology procedures. The quantification of the heme-sensor binding and the imaging studies will require the use spectroscopy/imaging techniques such as FLIM and FLIM-FRET.The project is under the supervision of Prof. Emma Raven at the University of Bristol and will involve the collaboration of Dr. Andrew Hudson (Leicester University) and the Wolfson Biomaging center (Bristol).

由于血红素金属磷脂的潜在毒性性质和疏水性，因此该假体跨细胞膜的必要性表明存在高度控制的递送机制。此外，累积有关血红素信号传导和调节特性的证据表明，这种无处不在的辅助因子比传统上想象的要宽得多。为了阐明细胞代谢和稳态中的血红素作用，需要对不稳定血红素池的生物学意义的新见解。作为对这一宏伟方案的贡献，该项目的目的是设计和构建一个传感器，以定量细胞中不稳定血红素浓度。这将涉及荧光标记的蛋白质的构建，这些蛋白可以在血红素浓度和条件范围内结合血红素。此外，将利用融合血红素结合蛋白的光学特性来进行成像，试图将血红素跟踪到细胞中。重组蛋白将主要在大肠杆菌中表达，并利用适当的工程质粒载体。纯化和表征将涉及标准分子生物学程序。血红素传感器结合和成像研究的量化将需要使用光谱/成像技术，例如FLIM和FLIM-FRET。该项目在布里斯托尔大学的Emma Raven教授的监督下，并将涉及Andrew Hudson（Leicester University）和Wolfson Biomaging Center（Bristol）的合作。