| Isolation and Purification of Viruses Infecting Cyanobacteria Using a Liquid Bioassay Approach
|
|
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。
|
|
|
| Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
|
|
Author:
Date:
2017-10-05
[Abstract] Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels.
[摘要] Hsp90，其伴侣伴侣Cdc37和激酶之间的相互作用已经在三十多年的生物化学研究中被证明在酵母与人类的生物体内在功能上是重要的。 然而，形成一个稳定的结构研究复合物是难以捉摸的。 在该方案中，我们描述了利用病毒2A序列以相似水平滴定三种蛋白质的来自酿酒酵母的Hsp90-Cdc37-Cdk4激酶蛋白复合物的表达和纯化。
【背景】Hsp90分子伴侣与其客体激酶之间的稳定形成复合物已经被证明在体外是难治性的。以前的工作表明，Hsp90的共伴伴Cdc37与昆虫Sf9细胞中的客体激酶的过表达导致Sf9 Hsp90，外源Cdc37和外源激酶（Vaughan等人，2006）之间的稳定复合物。然而，昆虫细胞培养需要特殊的设备，比其他研究较好的表达系统（如细菌和酵母）要难以进行遗传操作，并且显着较慢地生长和克隆。上述蛋白质在E中的共表达。大肠杆菌不产生可溶性激酶/稳定复合物。我们认为，酿酒酵母将具有必要的机制来帮助折叠和促进复合物的形成，并试图通过共同表达这些蛋白质来产生人Hsp90β，人Cdc37和人Cdk4激酶之间的复合物， S上。酵母。为了获得三种蛋白质的化学计量表达，我们利用病毒2A肽，其允许三个蛋白质在一个mRNA上转录，随后在翻译阶段切割。该系统已被用于人类细胞系和兔网状细胞（Kim等人，2011; ...
|
|
|
| In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
|
|
Author:
Date:
2017-09-20
[Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other proteins. We have previously established the mechanisms by which the polymerases ε and δ are targeted to their ‘correct’ strands, as well as quality control mechanisms that evict polymerases when they associate with an ‘incorrect’ strand. Here, we provide a practical guide to differentially assay leading and lagging strand replication in vitro using pure proteins.
[摘要] 真核生物复制品是重复DNA的多蛋白复合物。 复制品被雕刻成连续的前导链合成与不连续的滞后链合成，主要通过DNA聚合酶ε和δ以及解旋酶，聚合酶α-引发酶，DNA滑动夹，夹带载体和许多其它蛋白质进行。 我们以前已经建立了聚合酶ε和δ靶向其“正确”链的机制，以及在与“不正确”链相关联时驱赶聚合酶的质量控制机制。 在这里，我们提供了使用纯蛋白质在体外差异测定前导和滞后链复制的实用指南。
Using pure proteins from Saccharomyces cerevisiae, our lab was the first to reconstitute a functional eukaryotic DNA replisome, a ~2 MDa complex that includes the 11-subunit CMG helicase (complex of Cdc45, Mcm2-7, GINS heterotetramer), the 4-subunit DNA polymerase (Pol) ε, the 4-subunit Pol α-primase, the PCNA (Proliferating Cell Nuclear Antigen) clamp homotrimer ring shaped processivity factor that ...
|
|
|