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CHD4蛋白截短体融合蛋白的表达、纯化和鉴定

Expression,purification and identification of CHD4/Mi-2β truncated proteins
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摘要 目的构建CHD4/Mi-2β基因的截短体原核表达重组质粒,在大肠杆菌中诱导表达并对GST融合蛋白进行纯化和初步鉴定。方法采用PCR方法扩增CHD4/Mi-2β的染色质调节区(CHD4-C)、解螺旋酶模序(CHD4-H)及DNA结合区(CHD4-D)基因片段,将目的基因片段插入原核表达载体pGEX-5T构建带GST标签的重组质粒,阳性克隆行DNA序列测定。IPTG诱导GST-CHD4-C、GST-CHD4-D和GST-CHD4-H原核表达,谷胱甘肽琼脂糖珠纯化融合蛋白,并对融合蛋白Western blot鉴定。结果构建了3个CHD4/Mi-2β基因的截短体重组质粒;IPTG诱导显示,GST-CHD4-C、GST-CHD4-D和GST-CHD4-H融合蛋白主要以可溶性蛋白形式在细胞裂解液上清中表达,表达产物的相对分子质量分别为130、110、90ku。谷胱甘肽琼脂糖珠纯化融合蛋白,纯化产物的纯度最高可达94.5%。Western blot证实各融合蛋白与抗GST单克隆抗体发生特异性结合反应,分子质量与估计值相符,提示为GST融合表达的CHD4/Mi-2β截短体蛋白。结论成功纯化了较高纯度的GST-CHD4-C、GST-CHD4-D和GST-CHD4-H融合蛋白,为进一步研究CHD4蛋白在染色质重塑中的作用奠定了实验基础。 Objective To construct chromodomain helicase DNA-bingding protein 4 (CHD4/Mi-2β) truncated recombinant plasmids, induce the expression of GST fusion proteins in E. coli, and purify and identify GST fusion proteins of CHD4/Mi-2β. Methods The gene fragments of chromodomains (CHD4-C), SWI2/SNF2-related ATPase/helicase region (CHD4-H) and DNA-binding domain (CHD4-D) were amplified by PCR and cloned into pGEX-5T vector. After the target genes were sequenced, the plasmids were transformed into E. coli BL21 (DE3) respectively, and induced with 0.8 mmol/L IPTG to express fusion proteins. The GST-fusioned recombined proteins were purified with glutathione-agarose resin, and identified by SDS-PAGE and Western blotting. Results Three CHD4 truncated recombinant plasmids were constructed. The recombinant fusion proteins of GST-CHD4-C, GST- CHD4-D and GST-CHD4-H were expressed in E. coli BL21 (DE3) and existed mostly in the form of soluble protein with expected relative molecular weight of 130, 110 and 90 ku. The purity of the fusion proteins reached 94.5% after purification by high-affinity glutathione-agarose resin. Conclusion GST-CHD4-C, GST-CHD4-D andGST-CHD4-H have been successfully purified, which lays an experimental foundation for further study of CHD4/ Mi--2β in chromatin remodeling.
出处 《西安交通大学学报(医学版)》 CAS CSCD 北大核心 2014年第1期12-15,共4页 Journal of Xi’an Jiaotong University(Medical Sciences)
基金 国家自然科学基金资助项目(No.30960093 31160183 31270894)~~
关键词 CHD4 Mi-2β pGEX ST 融合蛋白 蛋白表达 纯化 CHD4/Mi-2β pGEX-5T fusion protein protein expression purification
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