Cloning, in vitro expression and functional assay of the gene encoding with a protein of the methyl-CpG binding domain
Date Issued
2005
Date
2005
Author(s)
Huang, I-Hua
DOI
zh-TW
Abstract
The methylation of DNA, in a wide range of species, has numerous biological roles involved in the cellular inheritance of biological traits. It is generally recognized that under certain conditions DNA methylation is often observed in GC-rich sequences called CpG islands (CGIs) that are frequently located at the 5’ regions of genes and often contain regulatory elements such as promoter sequences. Therefore, CGIs can be regarded as a useful markers for identified gene. When methylation at CpG islands does occur, it is known to paly an important role in gene silencing associated with genomic imprinting that has been implicated in regulating the early development of emryos. However, the mechanism and consequences of this unusual mode of gene regulation are not fully understood. Available techniques are essentially requested for the successful construction in a genome-wide of the CpG islands and it would be of great valuable for the further analysis of CpG island methylation, and this is particularly important with regard to the regulation of gene activity. Attempts of the present study were made to produce a protein equipped with the domain characterized by highly specificity of binding to the methyl-CpG sequences of gene(s) and it is anticipated that this protein may then provide for further application of genome-wide construction of the CpG islands.
To meet with the above purposes, experiments were first conducted to clone the gene encoding with a protein of the methyl-CpG binding domain (MBD). Using a genetic fusion molecular system, the MBD coding sequence was inserted into PinPoint Xa protein expression vector for construction of an in-frame Biotin-Tag fusion plasmid. Biotin tag-MBD recombinant protein was then expressed in a bacterial system. Results from these initial studies indicated that not only the expression level of the recombinant proteins was low but also a large proportion of the target proteins were appeared as a form of insoluble. However, instead of using a new protein expression vector named as pET-SUMO-PinPoint-MBD, it was found that both of the expression level and the solubility of the SUMO fusion recombinant protein were significantly improved. The 6xHis-SUMO-Biotin-MBD recombinant proteins, after purification by using affinity chromatography against to the nickel-chelating affinity resin, appeared to be capable of suffering from SUMO protease cleavage and removal of both the SUMO protease and the SUMO fusion recombinant proteins became much easier after the cleavage reaction and further subjected to affinity chromatography with the nickel-chelating affinity resin. Moreover, the resulted recombinant proteins, Biotin tag-MBD, were confirmed to be highly retaining of their methyl-CpG binding activity when they had been subjected to the electromobility shift assay (EMSA) for the determination of their bioactivity.
Subjects
甲基化CpG結合功能域
甲基化
methyl-CpG binding domain
methylation
Type
thesis
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