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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Author:
Date:
2018-06-05
[Abstract] The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis (Calabro et al., 2000).
[摘要] 糖胺聚糖透明质酸(HA)是细胞外基质的关键组分。 HA的分子量是不均匀的,可以达到几百万至几百道尔顿。 HA对细胞行为的影响与尺寸有关; 片段化的HA作为危险信号起作用,刺激细胞迁移和增殖并且是促炎性的,原生高分子量HA抑制炎症。 因此,在研究HA在组织稳态和病理学中的作用时,分析HA的大小分布非常重要。 该协议描述从小鼠乳腺分离HA,但也可以应用于其他组织。 分离的HA的质量足以通过凝胶电泳分析大小分布(Calabro等人,2000)。
【背景】糖胺聚糖HA由N-乙酰葡糖胺和β葡萄糖醛酸二糖组成,并且是细胞外基质的普遍存在的组分。 高分子量HA通过酶促降解和被活性氧和氮物质氧化而碎裂。 在健康组织中,大部分HA具有高分子量。 片段化HA的积累在病理过程中起着危险信号的作用(Tolg等人,2012和2017; Yuan等人,2015)。 例如,HA片段刺激炎症,而高分子量HA抑制炎症。 HA通过与细胞膜受体相互作用影响细胞行为,如细胞迁移和增殖,导致信号通路的激活。 由于受体-HA相互作用受HA大小的影响,HA对组织生物学的影响不仅取决于HA量,而且取决于各个细胞的HA大小分布和HA受体表达。
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