Expression and Ni-NTA-Agarose Purification of Recombinant Hepatitis C Virus E2 Ectodomain Produced in a Baculovirus Expression System
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Author:
Date:
2018-10-05
[Abstract] In this protocol, we describe the production and purification of the ectodomain of the E2661 envelope protein (amino acids 384-661) of the Hepatitis C virus, which plays a fundamental role in the entry of the virus into the host cell. This protein has been expressed in both prokaryotic and eukaryotic systems but in small quantities or without native protein characteristics. In our case, we use the Baculovirus expression system in insect cells. E2661 is secreted into the extracellular medium and purified by means of affinity chromatography a Ni-NTA-column because the protein has a tag of six histidines at its amino terminal end. The purified protein possesses a native-like conformation and it is produced in large quantities, around 5-6 mg per liter.
[摘要] 在该协议中,我们描述了丙型肝炎病毒的E2 661 包膜蛋白(氨基酸384-661)的胞外域的产生和纯化,其在病毒进入中起基础作用。 进入宿主细胞。 该蛋白质已经在原核和真核系统中表达,但是少量或没有天然蛋白质特征。 在我们的例子中,我们在昆虫细胞中使用杆状病毒表达系统。 E2 661 被分泌到细胞外培养基中并通过亲和层析Ni-NTA-柱纯化,因为该蛋白质在其氨基末端具有六个组氨酸的标签。 纯化的蛋白质具有天然样构象,并且大量生产,每升约5-6mg。 【背景】丙型肝炎病毒(HCV)是全世界慢性肝炎,肝硬化和肝细胞癌的主要原因(Major et al。,2001; Alter,2006)。此时,没有HCV疫苗,抗病毒药物用于治疗HCV感染(Imran et al。,2014)。然而,治疗费用昂贵且不是100%有效(Kohli et al。,2014)。 HCV包膜糖蛋白E2负责与细胞受体的相互作用,因此它是研究病毒感染周期的第一步的主要候选者。由于糖基化和聚集,先前的表达系统产生低水平的异质蛋白质,并且难以区分经历生产性和非生产性折叠的分子(Flint ...
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Direct Visualization of the Multicopy Chromosomes in Cyanobacterium Synechococcus elongatus PCC 7942
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Author:
Date:
2018-08-05
[Abstract] Cyanobacteria are prokaryotic organisms that carry out oxygenic photosynthesis. The fresh water cyanobacterium Synechococcus elongatus PCC 7942 is a model organism for the study of photosynthesis and gene regulation, and for biotechnological applications. Besides several freshwater cyanobacteria, S. elongatus 7942 also contains multiple chromosomal copies per cell at all stages of its cell cycle. Here, we describe a method for the direct visualization of multicopy chromosomes in S. elongatus 7942 by fluorescence in situ hybridization (FISH).
[摘要] 蓝细菌是进行含氧光合作用的原核生物。 淡水蓝藻 Synechococcus elongatus PCC 7942是用于研究光合作用和基因调控以及生物技术应用的模式生物。 除了几种淡水蓝藻, S. 细胞 7942在其细胞周期的所有阶段每个细胞还含有多个染色体拷贝。 在这里,我们描述了一种直接可视化 S中多拷贝染色体的方法。 通过荧光原位杂交(FISH)测定细菌 7942。
【背景】蓝藻是原核微生物,其利用与高等植物中的叶绿体类似的产氧光合系统。虽然 Escherichia coli 和枯草芽孢杆菌等细菌具有单个圆形染色体,但是几种淡水蓝细菌每个细胞有多个圆形染色体(Mann和Carr,1974; Labarre 等人,1989; Binder和Chisholm,1995)。淡水蓝藻 Synechococcus elongatus PCC 7942(以下简称 S.longatus 7942)每个细胞携带3-8个染色体拷贝(Griese et al。,2011; Watanabe et al。,2012; Watanabe et al。,2015)。已经建立了荧光报告子 - 操纵子系统来获取 S的多拷贝染色体的图像。 elongatus 7942(Chen et al。,2012; Jain et ...
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