| Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy
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Author:
Date:
2021-02-05
[Abstract] Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or binding of small molecules. Fourier transform infrared (FTIR) difference spectroscopy is a well-suited method to study the light-induced structural responses of photoreceptors including changes in cofactor, side chains and secondary structure. Here, we describe a protocol to study the response of cofactor and protein in living E. coli cells via in-cell infrared difference (ICIRD) spectroscopy using the attenuated total reflection (ATR) ...
[摘要] [摘要]最近开发了几种细胞内光谱技术，以研究蛋白质在其天然环境中的结构和机理。体内条件与为体外实验选择的条件大不相同。因此，细胞环境可例如通过分子拥挤或小分子结合来影响蛋白质机制。傅立叶TRANSF ORM红外（FTIR）光谱差异是非常适合的方法来研究的光诱导结构响应小号光感受器，包括辅因子的变化，侧链和二级结构。在这里，我们描述了一种协议，用于研究活大肠杆菌中辅因子和蛋白质的响应 通过使用衰减全反射（ATR）配置的细胞内红外差（ICIRD）光谱分析细胞。蛋白质在大肠杆菌中过表达，将细胞转移到盐溶液中，并使用荧光光谱法确定每个细胞的拷贝数。将悬浮液离心，并将浓缩的细胞转移到FTIR光谱仪内的ATR池中。该恒温细胞密封并从与LED的顶部照明。在照射之前和之后记录强度光谱，以产生活细胞内部受体的差异光谱。利用ICIRD光谱，可在近乎自然的环境中解决可溶性感光体的结构变化。该方法在环境条件下可在H 2 O中运行，无标记，对蛋白质大小没有任何限制，并且不需要任何纯化步骤。
图形摘要：
使用衰减全反射对活大肠杆菌中的感光细胞进行细胞内红外差异光谱分析。
[背景]光感受器在从细菌，真菌，藻类和植物到动物的各种生物体的光感测和光适应中起着至关重要的作用（Möglich等，2010 ; ...
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| Expression and Partial Purification of His-tagged Proteins in a Plant System
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Author:
Date:
2015-09-05
[Abstract] Plant protein expression can be a challenging enterprise in any biochemical or molecular biology research project. Several heterologous systems like bacteria, yeast, insect cells and cell free systems have been used to produce plant proteins for in vitro experiments and structural characterization. However, due to particularities of plant proteins, for example the specific type and abundance of post-translational modifications (e.g. glycosylation), a plant system to express plant proteins is extremely desirable. The use of Nicotiana benthamiana (N. benthamiana) plants for protein expression has proven to be quick and reliable. To illustrate the robustness and rapidity of this system, recent efforts to produce the first protein based drug against the ...
[摘要] 植物蛋白表达可以是任何生物化学或分子生物学研究项目中的挑战性企业。几种异源系统如细菌，酵母，昆虫细胞和无细胞系统已被用于生产植物蛋白用于体外实验和结构表征。然而，由于植物蛋白的特殊性，例如翻译后修饰（例如糖基化）的特异性类型和丰度，表达植物蛋白的植物系统是非常需要的。已经证明使用本氏烟草（本生烟草）植物进行蛋白质表达是快速和可靠的。为了说明该系统的稳健性和快速性，最近在N中进行了生产针对埃博拉病毒的第一种基于蛋白质的药物的努力。本草案蛋白质表达系统（Choi等人，2015）。
该方案描述了一种简单的系统，用于表达和富集（亲和纯化） > N。本生烟草叶，其被成功地用于拟南芥果胶乙酰酯酶，PAE8和PAE9的表征中（de Souza等人，2014）。
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