【作者】 王飞；

【导师】 饶子和； 杨海涛；

【作者基本信息】 天津大学 ， 生物学， 2017， 硕士

【摘要】 1962年下村修等人分离得到的绿色荧光蛋白及其各种突变体在生物科学研究中主要用于标记和示踪。但是绿色荧光蛋白发射光谱仅仅局限在440-529nm,细胞内成像时背景高,应用于活体生物的标记和示踪效果不够好。在1999年首次报道的红色荧光蛋白与绿色荧光蛋白相比其优点非常明显。而本论文中所研究的远红外红色荧光蛋白能够很好的解决生物体内成像背景高的问题。2016年Erik A Rodriguez等人用TeAPCα经过突变产生了符合要求远红外红色荧光蛋白smURFP。突变体smURFP不仅亮度高而且能够在642-670nm范围内激发荧光,满足用于生物体内研究的需求。如何了解该蛋白质发光的结构机制,并利用其生化特性改造、设计新一代远红外红色荧光蛋白具有重要的科学价值和应用前景。而本文将smURFP作为研究对象,通过分子克隆等手段成功构建了一系列克隆,并在大肠杆菌中实现了目的蛋白进行了过量表达。通过亲和层析、离子交换层析和凝胶过滤层析等手段对目的蛋白进行了纯化,并获得了纯度较高的smURFP蛋白。通过对结晶条件的筛选我们获得了smURFP蛋白结晶。经过pH优化、添加剂优化等过程最终得到高质量的蛋白质单晶,并在上海同步辐射光源进行数据收集。虽然母体smURFP蛋白晶体衍射达到2.1?,在结构解析过程中利用分子置换法仍无法确定晶体相位。为了解决相位问题,我们后续对smURFP蛋白进行硒代甲硫氨酸标记,拟采用单/多波长反常散射的方式确定相位。在smURFP蛋白中有4个甲硫氨酸(除去起始的甲硫氨酸),通过硒标蛋白的制备和纯化,我们得到了的纯度较高的硒代甲硫氨酸标记的目的蛋白,并已获得较好的晶体。以上对smURFP蛋白的初步晶体学研究,有助于我们为后续解析其蛋白结构、了解其发光机制、进一步改造该荧光蛋白奠定了基础。更多还原

【Abstract】 In 1962 Osamu Shimomura and his team found and isolated the green fluorescent protein from Aequorea victoria jellyfish.Green fluorescent protein and its various mutants are used in biomedical research primarily for labeling and tracing.However,since the emission spectrum of green fluorescent protein is limited to 440-529 nm,the background is high in intracellular imaging,and the labeling and tracer effect applied to living creatures are unsatisfactory.In 1999,red fluorescent protein was first reported,and it’s advantage was obvious compared with green fluorescent protein.And the far infrared red fluorescent protein studied in this paper can be a good solution to the problem of high imaging background in vivo.In 2016 Erik A Rodriguez et al.Produced a new far-infrared fluorescent protein named smURFP.The mutant smURFP not only has the highest brightness but also the ability to excite fluorescence in the range of 642-670 nm to meet the requirements for in vivo research.How to understand the structural mechanism of the protein luminescence,and use its biochemical characteristics of transformation,design a new generation of far infrared infrared fluorescent protein has important scientific value and application prospects.In this paper,smURFP was used as a research object,and a series of clones were successfully constructed by molecular cloning,then in E.coli the protein was expressed in large quantities.The smURFP protein with high purity was purified by affinity chromatography,ion exchange chromatography and gel filtration chromatography.The crystallization conditions of smURFP protein were screened to determine the appropriate crystallization buffer.After the pH optimization and additive optimization process,the protein crystals were obtained and the protein was sent to Shanghai Synchrotron radiation for data collection.Although the natural smURFP protein crystallizes to 2?,it is difficult to determine the crystal phase in the data analysis process by molecular replacement method.In order to solve the phase problem,we follow the subsequent smURFP protein selenomethionine labeling,intended to use multi-wavelength anomalous scattering to determine the phase.In the smURFP protein there are four methionine(in addition to the initial methionine),after cloning and purification of the expression of the selenium-labeled protein,we obtained the higher purity selenomethionine-labeled target protein.After the crystallization conditions of the screening and optimize the crystals we get better crystal.Above the preliminary study of smURFP protein will help us to lay the foundation for further analysis of its protein structure,to understand its luminescence mechanism,and to further transform the red fluorescent protein.更多还原