Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
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Author:
Date:
2021-02-20
[Abstract] In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using ...
[摘要] [摘要]在过去的几年中,作为GPCR-G复杂结构数量激增的证据,用于结构生物学的GPCR和G蛋白的表达已取得了巨大的成功,主要是在昆虫和哺乳动物细胞系统中,导致了370多个已解决了70多个GPCR的结构。但是,挑战仍然存在,特别是在构象转变和动力学研究领域,即使与X射线和冷冻EM相比(5 mg / ml,3μl /样品),也需要大量的受体和G蛋白。当应用NMR光谱法(5 mg / ml,250μl /样品)时。结果,i的表达水平 nsect和哺乳动物系统也很难满足这一需求,更不用说使用绝大多数系统使用这些系统生产GPCR和G蛋白的成本高昂了。因此,需要探索一种具有广泛适用性的有效,负担得起的实用方法。毕赤酵母表达系统已在GPCR制备中显示出其希望,并具有其他真核表达系统无法比拟的许多优点。在该系统中表达的GPCR价格便宜,易于操作,并且能够进行同位素标记。在此,我们提出最近开发并在我们的实验室升级的相关协议,包括表达和纯化的毕赤酵母衍生GPCR以G沿α和G βγ蛋白。我们预期这些协议将促进GPCR及其复合物的构象转变和动力学研究。
[背景] G蛋白偶联受体(GPCR)是最大的膜蛋白家族,在许多(病理)生理活动中起着关键作用。GPCR的或它们的效应物的功能障碍会导致各种病症,包括神经变性疾病,癌症,和慢性炎症(Overington等人,2006) ...
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Live Cell Measurement of the Intracellular pH of Yeast by Flow Cytometry Using a Genetically-Encoded Fluorescent Reporter
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Author:
Date:
2020-06-20
[Abstract] The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively low-throughput such that collecting enough data to fully characterize populations of cells is challenging. Flow cytometry avoids this drawback, and is a powerful tool that allows for rapid, high-throughput measurement of fluorescent readouts in individual cells. When combined with pH-sensitive fluorescent reporters, it can be used to characterize the intracellular pH of large populations of cells at the single-cell level. We adapted microscopy and ...
[摘要] [摘要 ] 酵母的细胞内PH是对生长状态和环境条件响应发生变化的严格调节的生理线索。荧光报告基因可响应局部PH的变化而改变荧光,可用于测量细胞内的PH。常被用来进行此类测量,它是相对低吞吐量,从而收集足够的数据来充分体现群体的细胞是一种挑战。˚F 低流式细胞术避免了这种弊病,而且是一个强大的工具,允许对于快速,高通量测量单个细胞中的荧光读数。 当与pH敏感荧光报告相结合,它可以被用来表征的细胞内pH 大人口小号在单细胞的细胞level.We适于显微镜和流式细胞术为基础的方法来测量酵母胞内pH值。细胞可以可以在接近自然的条件下生长直到测量点为止,并且该协议可以在变化的环境条件下适用于单点或动态(时间分辨)测量。
[背景 ] 酵母的细胞内pH值与像活力和生长速率特性相关,和细胞内pH的调节消耗的相当大的比例的细胞活力的资源(Orij 等人,2011) 。然而,细胞内pH值可迅速变化并且是高度对环境敏感,因此对于细胞生理学的这一重要方面,采用快速,最小扰动的测量方法至关重要。
将目标化合物的局部浓度转换为荧光读数的基因编码生物传感器彻底改变了我们表征细胞内环境的能力,对于某些传感器,绝对荧光强度与读数相关,这在细胞内进行时可能是一个问题测量,因为荧光依靠小号都在该传感器的表达水平,而略有差异小区到小区,和特征兴趣。[R ...
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Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome
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Author:
Date:
2016-11-20
[Abstract] Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.
[摘要] 由于其大尺寸,细菌致病性岛和其它连续操纵子可能难以使用常规方法克隆。在这里我们描述了一个强大的3步法从毒力质粒或粘粒转移大型定义的片段到更小的自主复制质粒或直接到细胞染色体,并入内源性酵母和λ红色同源重组系统的定义网站。该方法已经成功地用于分离和整合至少31kb的连续DNA,并且可以容易地适应于E的重组。大肠杆菌及其近亲。 [背景] 分离和繁殖大片DNA的能力大大扩展了基因网络和操纵子的研究。然而,用于该目的的传统使用的工程质粒,例如细菌人工染色体(BAC),虽然极其有用,但是受到DNA稳定性,拷贝数和复杂装配要求的问题的限制。或者,将构建体直接并入细菌染色体中通过减少由于存在多个基因拷贝引起的基因表达的变化以及确保基因的稳定维持而提供了优点,同时还避免了对抗生素选择的需要。这里描述的方法最初被设计为捕获和转移编码Shigella flexneri 3型分泌系统的31kb DNA操纵子到大肠杆菌染色体上(Reeves ...
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