New Methods for Site-Specific Protein Labelling

位点特异性蛋白质标记的新方法

• 批准号: RGPIN-2014-04702
• 负责人: Keillor, Jeffrey
• 金额: $ 4.95万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611194
• 关键词: New; Methods; Site; Specific; Protein

The human genome has been sequenced, allowing the identification of a huge number of putative genes. However, much work remains to be done to determine which genes are translated into proteins, where these proteins are located, and what biological roles they play, under different cellular conditions. To help address these critical fundamental questions, more powerful methods for monitoring the expression, localization and dynamic trafficking of specific proteins inside living cells are urgently required. Over the next funding period the Keillor research program will lead in an exciting new direction. We will address this urgent need by developing novel methods for the intracellular labelling of specific proteins, allowing their detection by fluorescence microscopy. The development of such methods will require a broad multidisciplinary approach that includes organic synthesis, physical organic studies and protein engineering. Our specific objectives comprise the development of two complementary approaches for protein labelling - one based on enzyme-mediated labelling, and the other relying on the selective fluorogenic reactivity of small molecules. In the enzyme–based approach, we will use protein engineering to develop a bacterial enzyme that can recognize a specific peptide sequence genetically fused to a protein of interest, expressed in a mammalian cell. The engineered enzyme will have the ability to incorporate small synthetic substrates into the target peptide tag sequence. In turn, these substrates will bear functional groups that allow for direct detection and imaging. Alternatively, the substrates may also be designed to undergo bioorthogonal reactions with other synthetic molecules that facilitate detection. In our complementary small molecule approach, labelling agents will be synthesised that become fluorescent upon their reaction with a different specific peptide tag, genetically encoded on the protein of interest. We will perform spectroscopic studies on these fluorogenic agents, to optimize the increase of their fluorescence upon their addition reaction. Kinetic studies will also be performed to guide the control of their reactivity, thereby ensuring the specificity of their fluorogenic addition reaction with the intended target peptide sequence, even in living cells. The breadth of our expertise heavily favours the feasibility of the proposed studies. Our repertoire of demonstrated methodology includes organic synthesis, kinetic and spectroscopic studies, molecular biology, protein expression, mammalian cell culture and fluorescence microscopy. Ultimately, we will apply our new labelling methods to the cell-based investigation of the conformation, mechanism and inhibition of enzymes such as the transglutaminases. We have studied the in vitro activity and inhibition of this class of enzymes for nearly 20 years; the new labelling techniques developed in the next phase of our research program will transform our powers of investigation, allowing us, and others, to pursue the study of enzymes on an entirely new, and highly relevant, cellular level.

人类基因组已经测序，可以识别大量的推定基因。然而，还有很多工作要做，以确定哪些基因被翻译成蛋白质，这些蛋白质位于何处，以及它们在不同的细胞条件下发挥什么样的生物学作用。为了帮助解决这些关键的基本问题，迫切需要更强大的方法来监测活细胞内特定蛋白质的表达，定位和动态运输。 在下一个资助期内，Keillor研究计划将引领一个令人兴奋的新方向。我们将通过开发用于细胞内标记特定蛋白质的新方法来解决这一迫切需求，从而通过荧光显微镜对其进行检测。这种方法的发展将需要广泛的多学科方法，包括有机合成，物理有机研究和蛋白质工程。我们的具体目标包括开发两种互补的蛋白质标记方法-一种基于酶介导的标记，另一种依赖于小分子的选择性荧光反应性。 在基于酶的方法中，我们将使用蛋白质工程来开发一种细菌酶，该细菌酶可以识别与哺乳动物细胞中表达的感兴趣的蛋白质遗传融合的特定肽序列。工程化酶将具有将小的合成底物掺入靶肽标签序列的能力。反过来，这些底物将带有允许直接检测和成像的官能团。或者，也可以将基底设计成与促进检测的其他合成分子进行生物正交反应。 在我们的互补小分子方法中，将合成标记剂，这些标记剂在与不同的特定肽标签反应时发出荧光，这些肽标签在感兴趣的蛋白质上遗传编码。我们将对这些荧光剂进行光谱研究，以优化它们在加成反应中荧光的增加。还将进行动力学研究以指导其反应性的控制，从而确保其与预期靶肽序列的荧光加成反应的特异性，即使在活细胞中也是如此。 我们专业知识的广度极大地支持了拟议研究的可行性。我们的方法包括有机合成，动力学和光谱研究，分子生物学，蛋白质表达，哺乳动物细胞培养和荧光显微镜。最终，我们将应用我们的新的标记方法，以细胞为基础的调查的构象，机制和抑制酶，如转氨酶。我们已经研究了近20年的体外活性和抑制这类酶;在我们的研究计划的下一阶段开发的新标记技术将改变我们的调查能力，使我们和其他人能够在全新的，高度相关的细胞水平上进行酶的研究。