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A novel method for purification of the endogenously expressed fission yeast Set2 complex

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Title: A novel method for purification of the endogenously expressed fission yeast Set2 complex
Authors: Suzuki, Shota Browse this author
Nagao, Koji Browse this author
Obuse, Chikashi Browse this author
Murakami, Yota Browse this author
Takahata, Shinya Browse this author
Keywords: Chromatin
SRI domain
RNA polymerase II
Issue Date: May-2014
Publisher: Elsevier
Journal Title: Protein Expression and Purification
Volume: 97
Start Page: 44
End Page: 49
Publisher DOI: 10.1016/j.pep.2014.02.005
PMID: 24583182
Abstract: Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8 mu m), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. (C) 2014 Elsevier Inc. All rights reserved.
Type: article (author version)
Appears in Collections:理学院・理学研究院 (Graduate School of Science / Faculty of Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 高畑 信也

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