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

• Suzuki, Shota

• Nagao, Koji

• Obuse, Chikashi

• Murakami, Yota

• Takahata, Shinya

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.

Journal

• Protein Expression and Purification

  Protein Expression and Purification 97 44-49, 2014-05

  Elsevier

Keywords

• Chromatin
• Set2
• H3K36
• SRI domain
• RNA polymerase II
• FIM

Details

• CRID: 1050282813992504448
• NII Article ID: 120005438258
• HANDLE: 2115/56352
• ISSN: 10465928
• Text Lang: en
• Article Type: journal article
• Data Source:
  • IRDB
  • CiNii Articles
  • KAKEN