title: A novel method for purification of the endogenously expressed fission yeast Set2 complex.
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 (53-0.8μm), 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.
authors.affiliation: Graduate School of Chemical Science and Engineering, Hokkaido University, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: Graduate School of Life Science, Hokkaido University, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nagao
authors.affiliation: Graduate School of Life Science, Hokkaido University, Japan.
authors.forename: Chikashi
authors.initials: C
authors.lastname: Obuse
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Japan.
authors.forename: Yota
authors.initials: Y
authors.lastname: Murakami
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Japan. Electronic address: shinya.takahata@mail.sci.hokudai.ac.jp.
authors.forename: Shinya
authors.initials: S
authors.lastname: Takahata
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005374:Filtration / Q000379:methods; D011495:Histone-Lysine N-Methyltransferase / Q000302:isolation & purification* / Q000378:metabolism*; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D012568:Schizosaccharomyces / Q000737:chemistry / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D029702:Schizosaccharomyces pombe Proteins; D011495:Histone-Lysine N-Methyltransferase; C527202:Set2 protein, S pombe
keywords: Chromatin; FIM; H3K36; RNA polymerase II; SRI domain; Set2