Expression, purification and characterization of methyl DNA binding protein from Bombyx mori.
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.
Uno
Tomohide
T
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan. uno_tom@yahoo.co.jp
Nomura
Yuka
Y
Nakamura
Masahiko
M
Nakao
Atsushi
A
Tajima
Shoji
S
Kanamaru
Kengo
K
Yamagata
Hiroshi
H
Iwanaga
Yousuke
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D026481:Blotting, Far-Western / Q000379:methods; D012831:Bombyx / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D020224:Expressed Sequence Tags / Q000737:chemistry; D006135:Gryllidae / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment / Q000662:veterinary
D004268:DNA-Binding Proteins; C115368:MBD2 protein; D011994:Recombinant Proteins; D011494:Protein Kinases