Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2

Yuichi Ichikawa, Wataru Kagawa, Kengo Saito, Yuji Chikashige, Tokuko Haraguchi, Yasushi Hiraoka, Hitoshi Kurumizaka

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    Abstract

    During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1-Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1-Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1-Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement. The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1-Bqt2 complex.

    Original languageEnglish
    Pages (from-to)207-213
    Number of pages7
    JournalProtein Expression and Purification
    Volume88
    Issue number2
    DOIs
    Publication statusPublished - 2013

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    Keywords

    • Co-expression
    • Protein-protein complex
    • Solubilization
    • SUMO tag
    • Telomere

    ASJC Scopus subject areas

    • Biotechnology

    Cite this

    Ichikawa, Y., Kagawa, W., Saito, K., Chikashige, Y., Haraguchi, T., Hiraoka, Y., & Kurumizaka, H. (2013). Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2. Protein Expression and Purification, 88(2), 207-213. https://doi.org/10.1016/j.pep.2013.01.006