Expression, purification, and characterization of methyl DNA binding protein from Bombyx mori

Abstract

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.Abbreviation:EMSAElectro Phoretic Mobility Shift Assay

Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni

“…For Acheta domesticus MBD2/3, no NCBI accession number could be located; therefore, the amino acid sequence for this family member was retrieved from Uno et al . [ 25 ]. The sequences were inspected for homology and the presence of conserved and semi-conserved residues.…”

DNA methylation and methyl-CpG binding proteins: developmental requirements and function

“…It is possible that DNA methylation only plays an auxiliary role, for example, during development; the 5-methylcytosine content of the Drosophila genome as assessed by liquid chromatography showed the highest signal in the early embryo (Lyko et al 2000 ).While the Drosophila genome appears to be largely depleted of DNA methylation, other insect species such as the cabbage moth Mamestra brassicae , the peach potato aphid Myzus persicae , and the mealy bug Planococcus citri seem to utilize DNA methylation (Field et al 2004 ). Also, significant amounts of DNA methylation along with a MBD2/3 homolog have been identified in the silkworm moth Bombyx mori (Patel and Gopinathan 1987 ; Uno et al 2005 ). A functional DNA methylation system containing both Dnmt1 and Dnmt3 as well as a functional ortholog of the mammalian MBD family has been described in the honeybee, Apis melifera (Wang et al 2006 ).…”

Computational approaches for understanding the evolution of DNA methylation in animals

“…The genome of silkworm, Bombyx mori, encodes homologs of dnmt1 and dnmt2, 22 which are able to bind to purified methyl-binding domain proteins. 23 A low level of genomic DNA methylation is reported in B. mori and a more complete sequencing of the B. mori genome may reveal the presence of dnmt3. 2 Recently completed genomes of the wasp Nasonia vitripennis and the variation (Fig.…”