Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads.
Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads.
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使用亚微米珠对与顺铂损伤的 DNA 结合的细胞蛋白进行全面分析和纯化。
DOI：
10.1021/bc015560h
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发表时间：
2002
影响因子：
4.7
通讯作者：
H. Okuno
中科院分区：
文献类型：
--
作者：
T. Tomohiro;Jun;C. Sawa;H. Nakura;S. Yoshida;M. Kodaka;Mamoru Hatakeyama;H. Kawaguchi;H. Handa;H. Okuno
利用苯乙烯和甲基丙烯酸缩水甘油酯（GMA）共聚合成的新型亚微米微珠，直接从HeLaS3细胞粗核提取物中纯化顺铂（CDDP）损伤的DNA结合蛋白，建立了一种高效亲和纯化技术。微珠显著减少了非特异性蛋白质在固体表面的吸附和洗脱体积，简化了处理程序。与CDDP反应后，将端粒重复序列（TTAGGG）(n)固定在表面，制备用于纯化的微球。至少有9个蛋白与CDDP-DNA明显具有较高的亲和力，并通过氨基酸序列分析鉴定出，包括HMGB（高迁移率基团）、hUBF（人上游结合因子）和Ku自身抗原，这些蛋白先前被报道为cddp损伤DNA结合蛋白的成分。
A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.