Uno Tomohide, Nomura Yuka, Nakamura Masahiko, Nakao Atsushi, Tajima Shoji, Kanamaru Kengo, Yamagata Hiroshi, Iwanaga Yousuke
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
J Insect Sci. 2005;5:8. doi: 10.1093/jis/5.1.8.
A cDNA clone encoding methyl DNA binding domain-containing protein (bMBD2/3) was obtained by homology searches using a Bombyx mori fat body cDNA library. The cDNA encoded a polypeptide with 249 amino acids sharing 54% similarity with the methyl DNA binding protein from Drosophila melanogaster. To characterize the biochemical properties of bMBD2/3, the clone was expressed in Escherichia coli as His-tagged protein. The recombinant protein was purified to homogeneity using Ni-NTA superflow resin and heparin agarose. The protein showed specific methyl DNA binding activity and was phosphorylated by protein kinase in vitro. Immunoblotting using the purified antibody indicated that bMBD2/3 was expressed in almost all tissues. Using west-western blotting analysis, some proteins that interact with bMBD2/3 were identified in the brain. This is the first report that insect MBD is phosphorylated and is present in adult tissues. These results suggest that bMBD2/3 plays important roles in the DNA methylation-specific transcription of Bombyx mori.
通过使用家蚕脂肪体cDNA文库进行同源性搜索，获得了一个编码含甲基DNA结合结构域蛋白（bMBD2/3）的cDNA克隆。该cDNA编码一个含有249个氨基酸的多肽，与黑腹果蝇的甲基DNA结合蛋白具有54%的相似性。为了表征bMBD2/3的生化特性，该克隆在大肠杆菌中作为His标签蛋白表达。重组蛋白使用Ni-NTA超流树脂和肝素琼脂糖纯化至同质。该蛋白显示出特异性的甲基DNA结合活性，并在体外被蛋白激酶磷酸化。使用纯化抗体进行的免疫印迹表明，bMBD2/3在几乎所有组织中都有表达。通过western-western印迹分析，在脑中鉴定出了一些与bMBD2/3相互作用的蛋白。这是关于昆虫MBD被磷酸化并存在于成虫组织中的首次报道。这些结果表明，bMBD2/3在家蚕DNA甲基化特异性转录中发挥重要作用。
