化学合成光控蛋白探针用于研究免疫细胞信号传导机制

发布时间：2018-05-26 14:57

本文选题：蛋白质化学合成 + 一锅法多片段缩合　；参考：《清华大学》2016年博士论文

【摘要】：蛋白质化学合成是人工获取生物技术难以获得的蛋白质的有效手段。它能够在原子精度层面实现对蛋白质的任意改造,提供定点翻译后修饰(甲基化,乙酰化,磷酸化,泛素化等)蛋白质,对不同物理刺激(光,磁等)响应的蛋白质探针,以及蛋白质药物用于生命科学和生物医学研究。特别地,化学合成光控蛋白质探针能够高时空分辨的控制蛋白质的活性,而被广泛应用于研究高度动态的生命过程,比如细胞的定向运动,细胞内的信号传导等。然而,蛋白质化学合成还需要进一步发展方法以提高蛋白质合成的效率,并进一步扩展化学合成的蛋白质在生命科学的应用。本论文将介绍作者在博士期间发展的高效率化学合成蛋白质的新方法,及化学合成光控蛋白质探针在研究免疫细胞信号传导机制的应用。蛋白质化学合成近期的一个重要的方法进展就是发展一锅法多肽片段连接技术,克服由分离纯化连接中间产物带来的产率低和耗时费力的问题,以提高蛋白质化学合成的效率。虽然一锅法三片段连接技术已经被建立,却没有有效的一锅法更多片段(比如四片段)的缩合策略。本论文作者发展了一种稳健而高效的,基于对pH值敏感的新型N端Cys保护基——三氟乙酰氨甲基(Tfacm)的一锅法四片段蛋白合成策略。利用该策略,本论文作者成功的实现了一锅法四片段合成模型蛋白Crambin和人源趋化因子hCCL21。基于Tfacm的一锅法四片段化学合成蛋白质的策略,为化学合成更大,更复杂的蛋白质提供一种有力的手段。在发展蛋白质化学合成方法的基础上,本论文作者发展了高时空分辨的系统研究免疫细胞定向运动和活化早期的信号传导过程。作者化学合成双光子光控的趋化因子探针(hCCL5**),通过体外双光子解笼锁(0.1μm2)及共聚焦显微镜单细胞成像技术,证明了PI3K在T细胞趋化运动过程中不决定方向的感知,而影响细胞的持续性运动。同时,作者在小鼠表皮和淋巴组织内首次实现了利用双光子激活的探针hCCL5**人为的控制T细胞的运动和分布。此外,作者化学合成了光控蛋白抗原探针(HEL-K_(96)NPE),实现了光掩蔽极强相互作用(20 pM结合力)和极大相互作用面(1800?2)的抗体(HyHEL-10)-抗原(HEL)相互作用。并通过全内反射荧光显微镜(TIRFM),实现了对B细胞活化早期B细胞受体微簇形成和钙离子震荡反应等信号传导的动力学测量。这些光控蛋白探针为进一步理解和调控免疫细胞的功能提供了高分辨的分子工具。
[Abstract]:Protein chemical synthesis is an effective method to obtain protein which is difficult to be obtained by biological technology. It can transform proteins arbitrarily at the atomic precision level, providing fixed-point post-translational modification (methylation, acetylation, phosphorylation, ubiquitin, etc.) proteins, protein probes that respond to different physical stimuli (light, magnetism, etc.). And protein drugs for life sciences and biomedical research. In particular, chemically synthesized photo-controlled protein probes can control the activity of proteins in high spatial and temporal resolution, and are widely used to study highly dynamic life processes, such as cell directional movement, intracellular signal transduction, and so on. However, protein chemical synthesis needs to be further developed to improve the efficiency of protein synthesis, and to further expand the application of chemically synthesized proteins in the life sciences. This paper will introduce a new method of high efficiency chemical synthesis of proteins developed by the author during his Ph.D. and the application of chemically synthesized photo-controlled protein probes in studying the mechanism of signal transduction in immune cells. One of the most important methods of protein chemical synthesis is the development of one-pot peptide fragment binding technology to overcome the problems of low yield and time-consuming caused by the separation and purification of ligated intermediate products in order to improve the efficiency of protein chemical synthesis. Although the one pot three fragment linking technique has been established, there is no effective condensation strategy for more fragments (e. G. Four fragments) in one pot method. In this paper, the authors have developed a stable and efficient one-pot four-fragment protein synthesis strategy based on a novel N-terminal Cys protection group, trifluoroacetamethylammonium trifluoroacetylmethyl (Tfacm), which is sensitive to pH value. Using this strategy, the author successfully implemented the one-pot four-fragment synthesis of model protein Crambin and human chemokine hCCL21. The strategy of one-pot four-fragment chemical synthesis of proteins based on Tfacm provides a powerful means for chemical synthesis of larger and more complex proteins. On the basis of the development of protein chemical synthesis methods, the authors have developed a high space-time resolution system to study the early signal transduction processes of immune cells during their directional movement and activation. The authors have synthesized a chemically controlled chemokine probe hCCL5, using in vitro two-photon cage locking (0.1 渭 m ~ 2) and confocal microscope single cell imaging technique. The results show that PI3K does not determine the direction of T cell chemotaxis. And affects the cell's sustained movement. At the same time, in mouse epidermis and lymphoid tissues, the authors have for the first time realized the artificial control of T cell movement and distribution by using a two-photon activated probe hCCL5 *. In addition, the authors have chemically synthesized a photo-controlled protein antigen probe, HEL-KH _ (96) NPE, which realizes the interaction of the highly photomasking interaction (20 pm) and the maximal interaction surface (1800 ~ (2) with the antibody HyHEL-10 (antigen-hele). The kinetic measurements of signal transduction such as the formation of B cell receptor microclusters and Ca 2 + oscillation reaction in the early stage of B cell activation were carried out by means of total internal reflection fluorescence microscope (TIR). These light-controlled protein probes provide a high-resolution molecular tool for further understanding and regulating the function of immune cells.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392;O657.3

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