| Isolation and Purification of Viruses Infecting Cyanobacteria Using a Liquid Bioassay Approach
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Author:
Date:
2018-01-20
[Abstract] The following protocol describes the isolation and purification of viruses infecting cyanobacteria using a liquid bioassay approach. Viruses infecting cyanobacteria are also known as cyanophages. This protocol was written specifically for the isolation of cyanophages infecting freshwater cyanobacteria particularly, cyanobacteria that cannot be cultured on solid media. The use of a clonal cyanobacterial culture is recommended for the isolation of viruses. Growth conditions (i.e., media, light cycle and temperature) should be modified based on the host of interest.
[摘要] 以下方案描述了使用液体生物测定方法分离和纯化感染蓝细菌的病毒。 感染蓝细菌的病毒也被称为噬藻体。 本协议是专门为分离感染淡水蓝藻的蓝藻，特别是不能在固体培养基上培养的蓝细菌而编写的。 推荐使用克隆蓝藻培养物来分离病毒。 生长条件（即，介质，光周期和温度）应根据感兴趣的主体进行修改。
【背景】蓝藻是海洋和淡水系统中重要的营养生物。作为其他的水生微生物，蓝藻受到病毒感染（Suttle，2000）。例如，在海洋沿海地区，感染聚球蓝细菌的病毒滴度。 （Suttle和Chan，1993; Waterbury和Valois，1993），可以达到10-5 ml-1，并且基于温度，盐度和宿主丰度而不同。尽管蓝细菌及其病毒（也称为“蓝藻”）具有生态重要性，但只有少数病毒已经从有限的蓝藻菌株中分离出来。因此，通过筛选新的蓝藻菌株来分离新病毒是非常有意义的。以下议定书对于海洋和淡水系统都是相关的，但下面的例子将重点讨论分离和纯化感染淡水蓝藻的病毒（Chénardet al。，2015）。液体生物测定方法优于使用固体基质的公开方案的优点是可以靶向无法耐受噬菌斑测定方法经常使用的较高温度或不能在固体培养基上生长的蓝细菌。
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| Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example
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Author:
Date:
2017-09-05
[Abstract] The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating ...
[摘要] 与共激活因子p300的转录因子缺氧诱导因子（HIF）复合物，激活缺氧反应途径并允许肿瘤生长。每个相应蛋白质的CH1和CAD结构域形成p300和HIF之间的界面。正在开发靶向和抑制HIF / p300复合物形成的小分子化合物，目的是减少肿瘤生长。研究配体与p300-CH1相互作用的高分辨NMR光谱是必要的，为了进行结构和生物物理学研究，需要净化大量正确折叠的p300-CH1。 p300-CH1具有3个锌指和9个半胱氨酸残基，构成与试剂相容性和蛋白氧化相关的挑战。已经开发了一种通过在表达过程中并入锌并简化纯化时间来克服这些问题的方案，导致适合于结构NMR研究的最佳折叠蛋白质（120mg / 4L表达介质）的高产率。已证实最终重组p300-CH1的结构完整性是使用一维1 H NMR光谱和圆二色性最优的。该方案适用于纯化其他含锌指蛋白质。
【背景】由于不适当的血管灌注，实体瘤的发展与缺氧区的发展有关。对于缺氧微环境，肿瘤细胞过表达低氧诱导因子（HIF），一种异二聚体转录因子家族（Semenza，2002; Brat和Van Meir，2004; Kaur等，2005）。 HIFs结合p300（一种转录共激活因子），形成诱导HIF靶基因的复合物，从而激活缺氧反应途径并促进肿瘤生长（Kasper and Brindle，2006; Liu，2008）。涉及HIF / p300蛋白 ...
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| Purification of HCV-remodeled and Control ER Membranes
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Author:
Date:
2014-05-20
[Abstract] As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate HCV-remodeled host cell membranes and endoplasmic reticulum (ER) membranes of naïve cells, by using a functional NS4B HA-tagged subgenomic replicon and a C-terminally HA-tagged calnexin-overexpressing cell line, respectively. Post-nuclear whole cell membrane fractions are first enriched by density gradient centrifugation, followed by HA-specific affinity tag purification. Upon elution under native conditions, purified samples can be subject to a variety of ...
[摘要] 至于所有正链RNA病毒，丙型肝炎病毒（HCV）RNA复制与称为病毒复制工厂的重排宿主细胞膜紧密相关。 然而，到目前为止对病毒复制工厂的病毒和细胞成分了解甚少。 在这里，我们描述一个协议，通过使用功能NS4B HA标记亚基因组复制子和C末端HA标记calnexin过表达细胞系，特异性隔离HCV重塑的宿主细胞膜和内质网（ER）膜的幼稚细胞， 分别。 首先通过密度梯度离心富集核后全细胞膜级分，随后通过HA特异性亲和标签纯化。 在天然条件下洗脱时，纯化的样品可进行多种生物化学和功能测定。
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