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，是标记和可视化体内蛋白质的宝贵工具。基因编码蛋白质标签的能力与GFP结合小分子荧光团的优越特性将显著改善蛋白质的体内标记和可视化。本研究旨在利用Mtx和DHFR之间的高亲和力相互作用，将感兴趣的蛋白质与DHFR融合，然后用小分子Mtx偶联物标记蛋白质，从而在体内标记蛋白质。在初步结果中，我们展示了最近提交发表的工作，证明了这种方法的可行性，在中国仓鼠卵巢（CHO）细胞中标记了用Mtx-Texas红融合DHFR的质膜和核蛋白。然后，我们概述了我们在Aim 1中通过蛋白质工程开发正交Mtx-DHFR变体的计划。在Aim 2中，模块化Mtx-DHFR标记方法被应用于CALl，以区分定位于细胞后部的肌球蛋白-llb和定位于片层的肌球蛋白-llb在细胞运动中的作用，这是目前标记方法难以实现的实验。最后，在目标3中，我们演示了Mtx-DHFR标签在多色标记和FRET应用中的使用，并描述了与GFP相比这些标签的功效。

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.