| Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein – DNA Interactions in Magnaporthe oryzae
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Author:
Date:
2015-11-05
[Abstract] Chromatin immunoprecipitation (ChIP) is a powerful technology for analyzing protein-DNA interactions in cells. Robust ChIP procedures have been established for investigating direct interactions between protein and DNA. However, detecting indirect protein-DNA interactions in vivo is challenging. Recently, we used ChIP to analyze an indirect protein-DNA interaction between a putative histone demethylase, MoJmjC, and the promoter of the superoxide dismutase 1-encoding gene MoSOD1 in the rice blast fungus Magnaporthe oryzae (M. oryzae) (Fernandez et al., 2014). We tagged MoJmjC with the 3x FLAG epitope (Fernandez et al., 2014), instead of the larger and more commonly used GFP epitope, to mitigate against steric hindrance. We also employed ...
[摘要] 染色质免疫沉淀（ChIP）是一种强大的技术，用于分析细胞中的蛋白质-DNA相互作用。已建立了稳健的ChIP程序用于研究蛋白质和DNA之间的直接相互作用。然而，在体内检测间接蛋白-DNA相互作用是具有挑战性的。最近，我们使用ChIP来分析推定的组蛋白去甲基化酶MoJmjC和在稻瘟病真菌Magnaporthe oryzae中超氧化物歧化酶1编码基因MoSOD1的启动子之间的间接蛋白质-DNA相互作用（ oryzae ）（Fernandez ，，2014）。我们用3x FLAG表位标记MoJmjC（Fernandez等人，2014），而不是更大和更常用的GFP表位，以减轻空间位阻。我们还采用了使用DSG和甲醛的两步交联策略，而不是更常用于分析直接蛋白质-DNA相互作用的一步甲醛交联方法，以便更好地捕获间接的MoJm - MoSOD1 DNA相互作用。此外，我们已经表明两步交联适用于GATA转录因子，Asd4及其同源结合位点之间的直接蛋白-DNA相互作用的ChIP分析（Marroquin-Guzman和Wilson，2015）。在这里，我们提供详细的协议的染色质免疫沉淀，与通用的两步交联，在M。 oryzae 。
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| Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans
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Author:
Date:
2014-09-05
[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAG-tagged GfsA produced a ...
[摘要] GfsA是涉及O - 聚糖的生物合成的真菌β-半乳糖呋喃糖基转移酶。 为了研究GfsA的酶功能，我们尝试从两种异源宿主生物体获得该酶的重组蛋白。 然而，GfsA不能在大肠杆菌（大肠杆菌）或酿酒酵母（Saccharomyces cerevisiae）中表达为重组蛋白质（ cerevisiae ）。 因此，我们决定使用构巢曲霉（ A。nidulans ）作为宿主生物体，并产生使用染色体标签表达3×FLAG标记的GfsA的菌株。 为了证实其表达，从标记的菌株制备溶解的蛋白并用抗FLAG抗体分析。 表达3×FLAG标记的GfsA的菌株产生质量为约67kDa的功能性蛋白质。 该手稿中描述的方法允许纯化在A中表达的GfsA-3xFLAG蛋白。 构巢细胞。
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