| 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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| Adoptive Transfer of Memory B Cells
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Author:
Date:
2015-08-20
[Abstract] The adoptive transfer of antigen-specific B cells into mice that cannot recognize that specific antigen has two main advantages. The first is determining exactly when the B cells were transferred and exposed to antigen. The second is that all B cells that can bind that antigen are the ones that were transferred; no new antigen-specific B cells will emerge from the bone marrow. Thus all B cells that were exposed to the antigen and still alive after at least 4 weeks (8 weeks or more is ideal), are memory B cells.
Splenic B cells from B1-8 mice were prepared with an EasySep Mouse B Cell Enrichment Kit according to the manufacturer’s protocol. Single-cell suspensions were transferred intravenously into tail veins of recipient mice. Approximately 1 million NP+ B cells were ...
[摘要] 将抗原特异性B细胞过继转移到不能识别该特异性抗原的小鼠中具有两个主要优点。 第一种是确切地确定B细胞何时转移并暴露于抗原。 第二是可以结合该抗原的所有B细胞是转移的; 从骨髓中不会出现新的抗原特异性B细胞。 因此，暴露于抗原并在至少4周（8周或更长时间是理想的）后仍存活的所有B细胞是记忆B细胞。
根据制造商的方案用EasySep小鼠B细胞富集试剂盒制备来自B1-8小鼠的脾B细胞。 将单细胞悬浮液静脉内转移到受体小鼠的尾静脉中。 每只小鼠转移约100万个NP + B细胞。 转移后约12-24小时，用在明矾中沉淀的50μgNP-CGG腹膜内免疫小鼠。
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| Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves
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Author:
Date:
2015-06-20
[Abstract] Virus induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterisation. This method exploits a dsRNA-mediated antiviral defence mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid time frame as compared to the generation of stable transformants. Here we describe a simple method for silencing a target gene in barley seedling leaves using vectors based on the Barley Stripe Mosaic Virus (BSMV).
[摘要] 病毒诱导的基因沉默（VIGS）是基因功能表征的最有效的反向遗传学技术之一。 这种方法利用dsRNA介导的抗病毒防御机制在植物中。 与稳定转化体的产生相比，使用该方法允许研究人员在相对快的时间框架中产生快速表型数据。 在这里我们介绍了一种简单的方法，使用基于大麦条纹花叶病毒（BSMV）的载体，在大麦幼苗叶片中沉默目标基因。
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