Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

Methyl-CpG-binding domain （MBD） proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and SNAP-MBD2b, in E. coli. An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin. The engineered proteins purified by this method exhibited more than 93% purity and high binding avidity. We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis. However, this could be prevented by the use of 0.3 mol/L NaCI. The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E. coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization. The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.

一种快速高效的全基因组甲基化CpG岛富集方法的建立

高甲基化的CpG岛所致基因表观遗传学转录失活已经成为肿瘤表观基因组学研究的重要内容,但尚未建立一种能快速在全基因组水平上进行甲基化CpG岛的富集方法。利用甲基化结合蛋白MBD2b具有特异性结合甲基化DNA的特性,建立了一种基于DNA免疫共沉淀技术的全基因组甲基化CpG岛的富集方法。在大肠杆菌中表达重组的GST-MBD2b蛋白,通过Glutathione Sepharose 4B对重组蛋白进行纯化,制备成亲和层析柱,利用在不同的盐离子强度下甲基化DNA和非甲基化DNA的结合能力不同,对甲基化DNA进行富集。用甲基化酶SssI处理过的DNA片段与非甲基化DNA片段进行富条件的摸索,发现0.5mol/L KCl的浓度是甲基化DNA片段和非甲基化DNA片段得以分开的临界条件。样品的富集效率用Real Time PCR进行检测。结果表明,这种方法能够实现对全基因组甲基化DNA的有效富集且最高的富集倍数可达到100多倍。富集到的甲基化DNA可以进行后续的定量PCR,DNA测序和全基因组芯片的分析等工作,为大规模分析全基因组CpG岛甲基化的改变奠定了基础。