Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2
Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2
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DOI：
10.1016/j.pep.2013.01.006
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发表时间：
2013-04-01
影响因子：
1.6
通讯作者：
Kurumizaka, Hitoshi
中科院分区：
文献类型：
--
作者：
Ichikawa, Yuichi;Kagawa, Wataru;Kurumizaka, Hitoshi
在减数分裂过程中，染色体采取花束排列，这在真核生物中广泛保守。这种排列被认为在减数分裂的正常进行中起重要作用，通过介导同源染色体的正确配对。在粟酒裂殖酵母中，Bqt 1和Bqt 2的复合体在减数分裂前期早期的端粒聚集和随后的染色体花束排列中起着关键作用。Bqt 1和Bqt 2是介导端粒与核膜连接的多蛋白复合物的一部分。然而，复杂的结构细节需要澄清端粒聚类的机制。为了能够对Bqt 1和Bqt 2进行生物物理学研究，我们建立了一种分离纯化裂殖酵母Bqt 1-Bqt 2复合物的方法，该复合物与S.粟酒Bqt 1-Bqt 2复合体。一个共表达载体，其中一个表达的亚基融合到一个可移动的SUMO标签，产生了大量的蛋白质的可溶性部分。通过在纯化缓冲液中包括CHAPS（一种非变性两性离子去污剂）来保持去除SUMO标签后Bqt 1-Bqt 2复合物的溶解度。这些步骤使我们能够快速纯化稳定的Bqt 1-Bqt 2复合物。共纯化的Bqt 1和Bqt 2蛋白形成了一个稳定的异源二聚体，与体内研究的结果一致，显示了这两种蛋白的花束安排的要求。这里建立的表达和纯化程序将有助于进一步的生物物理研究的Bqt 1-Bqt 2复合物。(C)2013爱思唯尔有限公司保留所有权利。
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. (C) 2013 Elsevier Ltd. All rights reserved.