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標題: | 甲基化CpG結合功能域蛋白質之核酸序列選殖及其蛋白質之體外表現與功能分析 Cloning, in vitro expression and functional assay of the gene encoding with a protein of the methyl-CpG binding domain |
作者: | I-Hua Huang 黃怡華 |
指導教授: | 鄭登貴 |
關鍵字: | 甲基化CpG結合功能域,甲基化, methyl-CpG binding domain,methylation, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 在眾多的物種中,核酸甲基化在細胞生物特性之傳承上扮演許多生物學上的角色。一般認定在某些狀況之下,DNA甲基化通常會發生在富含GC(GC-rich)的序列,即CpG島(CpG island, CGI)中,這種CGI序列通常位於基因的5’端區域,而且常涵蓋有調節元素,例如:啟動子(promoter)序列。因此,CGI被認定為是一鑑定基因的有用標記(marker)。已知當CpG島發生甲基化之現象時,其在調控早期胚胎發育相關的銘印基因(imprinted gene)之靜默(silencing)作用上扮演很重要的角色。然而,目前對於這種獨特之基因調控模式其機制和重要性並不甚瞭解,因此需要一可分離基因組CpG島的技術,以期可以更進一步應用於分析調控基因活性之CpG島之甲基化狀態。因此,本研究乃嘗試生產可與甲基化CpG核酸序列產生高度特異之結合反應的甲基化CpG結合功能域(MBD)蛋白質,冀望未來能將此蛋白質應用於建立基因組CGI庫。
為達上述之研究目的,本試驗首先選殖出甲基化CpG結合功能域(MBD)之核酸序列,並運用基因融合分子的技術將MBD之核酸序列構築於PinPoint Xa蛋白質表現載體中,再於大腸桿菌表現Biotin tag- MBD重組蛋白質,由初步誘導蛋白質表現的結果顯示,不僅MBD重組蛋白質之表現水平低,而且大部分的標的蛋白質都以不可溶的形式存在。因此,再構築另一新的蛋白質表現載體,將其命名為pET- SUMO-PinPoint-MBD,透過此載體所表現之SUMO融合重組蛋白質,可以明顯改善並增加重組蛋白質之表現水平以及加強其溶解度。經運用鎳金屬螫合親和層析法純化的重組蛋白質6xHis-SUMO-Biotin -MBD,以SUMO蛋白 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35594 |
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