| 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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| Preparation of Synaptoneurosomes to Study the Synapse in the Murine Cerebral Cortex
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Author:
Date:
2021-01-20
[Abstract] The synapse is a complex structure where the transmission of information takes place. Synaptic dysfunction is one of the earliest pathophysiological events in several diseases, such as traumatic brain injury, cerebral ischemia, and neurodegenerative diseases. Thus, a methodology to study synaptic structure and function is crucial for the development of potential strategies for the treatment of many neurological diseases. Synaptoneurosomes (SNs) are structures assembled by the sealed presynaptic bouton and the attached post-synaptic density. Despite the fact that for a long time it has been recognized that SNs are a powerful tool to study synaptic function, composition, and structure, its use has been limited by the requirement of relatively large amounts of material to successfully ...
[摘要] [摘要]突触是一个复杂的结构,在此结构中发生信息的传递。小号ynaptic功能障碍是几种疾病,如脑外伤,脑缺血最早的病理生理活动之一,和神经退行性疾病。因此,研究突触结构和功能的方法学对于开发治疗许多神经系统疾病的潜在策略至关重要。突触神经小体(SN)是由密封的突触前钮扣和附加的突触后密度组装而成的结构。尽管很长一段时间以来,人们已经认识到SN是研究突触功能,组成的强大工具,在结构和结构上,其使用受到成功地隔离它们所需的相对大量材料的限制。在这里,我们描述了在低渗条件下执行的三步离心程序,以从少量大脑皮层中分离出SN。
图形摘要:
突触神经体的制备流程图。
[背景]突触是由连接至突触后终端和突触前布顿组装的结构所包围由星形细胞伸长率(Halassa等人,2007 )。在过去的十年中,见证了许多用于研究其结构和功能的高度复杂的技术的发展。但是,SN仍被大量研究者所使用,因为它们相对容易分离并产生有关突触结构和功能的有价值的信息。SNs的制备是在特定的低渗条件下进行的改良的细胞分离程序,可分离突触前突突和附着的突触后元件。在鉴定的最初阶段,它们被称为无细胞响应制剂,因为它们对激素具有响应(Chasin等,197 4 ;Horn和Phillipson ,1976 ...
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| Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus
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Author:
Date:
2020-09-05
[Abstract] The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus.
We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows ...
[摘要] [摘要 ] 单独基于CRISPR靶序列仍难以预测单个CRISPR / Cas9-sgRNA的切割效率。不同的细胞内环境(例如,取决于细胞类型或细胞周期状态)可能会通过DNA修饰(例如表观遗传标记和DNA结合蛋白(例如组蛋白))和染色质状态的改变来改变基因组DNA的可及性,从而影响sgRNA效率。核内基因组DNA的标记。
我们recen TLY 报道的多步骤方法筛选用于有效sgRNAs靶向的识别的单纯疱疹病毒(HSV-1)的基因组,并报告为在潜对裂解病毒相抑制CRISPR-Cas9的差动机构。该协议中详述的筛选平台允许在无细胞系统中以及在病毒靶细胞(例如人包皮成纤维细胞)的背景下逐步测试切割效率,然后对CRISPR / sgRNA的作用进行功能测试病毒蛋白的表达,复制,和再活化。该策略可以容易地应用于其他靶细胞,例如多能干细胞衍生的人感觉神经元或其他人DNA病毒。
背景 ] 单纯疱疹病毒(HSV)是的一种嗜神经性DNA病毒疱疹病毒科家族引起终身和无法治愈感染在大多数人群的,并可能导致显著发病率和死亡率(Liesegang环等人,1989; Roizman 等人。,2013) ...
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