Ichikawa Yuichi, Kagawa Wataru, Saito Kengo, Chikashige Yuji, Haraguchi Tokuko, Hiraoka Yasushi, Kurumizaka Hitoshi
Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162 8480, Japan.
Protein Expr Purif. 2013 Apr;88(2):207-13. doi: 10.1016/j.pep.2013.01.006. Epub 2013 Jan 19.
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.
在减数分裂过程中，染色体呈花束状排列，这种排列在真核生物中广泛保守。人们认为这种排列通过介导同源染色体的正确配对，在减数分裂的正常进程中发挥重要作用。在粟酒裂殖酵母中，Bqt1和Bqt2复合物在减数分裂前期早期的端粒聚集以及随后的染色体花束状排列中起关键作用。Bqt1和Bqt2是介导端粒与核膜附着的多蛋白复合物的一部分。然而，需要该复合物的结构细节来阐明端粒聚集的机制。为了能够对Bqt1和Bqt2进行生物物理研究，我们建立了日本裂殖酵母Bqt1 - Bqt2复合物的纯化程序，该复合物与粟酒裂殖酵母的Bqt1 - Bqt2复合物密切相关。一种共表达载体，其中一个表达的亚基与一个可去除的SUMO标签融合，在可溶性部分产生了大量的蛋白质。通过在纯化缓冲液中加入CHAPS（一种非变性两性离子去污剂），去除SUMO标签后Bqt1 - Bqt2复合物的溶解度得以维持。这些程序使我们能够快速纯化稳定的Bqt1 - Bqt2复合物。共纯化的Bqt1和Bqt2蛋白形成了稳定的异二聚体，这与体内研究结果一致，体内研究表明两种蛋白对于花束状排列都是必需的。这里建立的表达和纯化程序将有助于对Bqt1 - Bqt2复合物进行进一步的生物物理研究。
