Methotrexate Conjugates: A Molecular In Vivo Protein Tag

描述（由申请人提供）：用荧光取代基标记蛋白质，最常用的是使用绿色荧光蛋白（GFP）及其衍生物，是体内标记和可视化蛋白质的宝贵工具。与小分子荧光团的上级特性相结合的GFP遗传编码蛋白质标记的能力将显著改善蛋白质的体内标记和可视化。该提案旨在利用Mtx和DHFR之间的高亲和力相互作用，通过将感兴趣的蛋白质与DHFR融合，然后用小分子Mtx缀合物标记蛋白质来标记体内蛋白质。在初步结果中，我们目前的工作最近提交出版证明这种方法的可行性，标记质膜和核蛋白融合DHFR与MTX-德克萨斯红在中国卵巢（CHO）细胞。然后，我们概述了我们通过Aim 1中的蛋白质工程开发正交Mtx-DHFR变体的计划。在目的2中，将模块化Mtx-DHFR标记方法应用于CAL 1以区分定位于细胞后部的肌球蛋白-IIb与定位于细胞运动性中的板层的肌球蛋白-IIb之间的作用，这是用当前标记方法难以实现的实验。最后，在目标3中，我们展示了Mtx-DHFR标签用于荧光标记和FRET应用的用途，并表征了这些标签与GFP相比的功效。

DESCRIPTION (provided by applicant): The labeling of proteins with fluorescent substituents, most commonly done using green fluorescent protein (GFP) and its derivatives is an invaluable tool for tagging and visualizing proteins in vivo. The ability to genetically encode protein labels as with GFP combined with the superior characteristics of small molecule fluorophores will significantly improve the in vivo tagging and visualization of proteins. This proposal aims to exploit the high-affinity interaction between Mtx and DHFR to label proteins in vivo by fusing the protein of interest to DHFR and then labeling the protein with small-molecule Mtx conjugates. In Preliminary Results, we present work recently submitted for publication demonstrating the feasibility of this approach, labeling both a plasma membrane and a nuclear protein fused to DHFR with Mtx-Texas Red in Chinese Hamster Ovary (CHO) cells. We then outline our plans to develop orthogonal Mtx-DHFR variants via protein engineering in Aim 1. In Aim 2, the modular Mtx-DHFR labeling approach is applied to CALl to distinguish between the role of myosin-llb localized to the cell posterior to that localized to the lamella in cell motility, an experiment difficult to achieve with current labeling methodologies. Finally, in Aim 3 we demonstrate the use of Mtx-DHFR labels for multicolor tagging and FRET applications and characterize the efficacy of these labels in comparison to GFP.