摘要
目的:研究获得N端/C端融合组蛋白H3K4M小肽的EML3融合蛋白的方法。方法:将组蛋白H3(aa1~15)小肽编码区融合进表达EML3 Agenet结构域重组质粒中,得到N端/C端融合表达H3K4M的质粒,转化至大肠杆菌BL21(DE3)中。经扩大培养,加入IPTG后在15℃条件下诱导6×His标签EML3融合蛋白的表达。经镍柱亲和层析及凝胶过滤层析进行纯化后,通过SDS-PAGE和考马斯亮蓝染色检测蛋白的纯度。结果:通过质粒单点突变、质粒酶切、PCR克隆以及同源重组连接,成功构建EML3 N/C端融合组蛋白H3K4M的重组质粒,测序证实序列正确,而且重组质粒可在大肠杆菌中成功诱导融合蛋白的表达。其中EML3C端融合组蛋白H3K4M可通过凝血酶酶切从而得到游离的H3K4M小肽以及EML3 Agenet结构域蛋白。结论:成功构建EML3融合组蛋白H3K4M重组质粒,并诱导表达及纯化得到高纯度的EML3融合蛋白。
This paper aims to develop a method for obtaining EML3 fusion protein containing N-terminal or C-terminal fusion with histone H3K4M peptide.The encoding region of H3 peptide(aa 1-15) was fused into the recombinant plasmid expressing EML3 Agenet domain,resulting in the fusion plasmid expressing H3K4M fusion protein at N-or C-terminals.The plasmid was then transformed into Escherichia coli BL21(DE3).After expansion culture,the expression of the 6×His-tagged EML3 fusion protein was induced at 15℃ by adding IPTG.The purity of the protein was assessed by SDS-PAGE and coomassie brilliant blue staining,after purification by nickel affinity chromatography and gel filtration chromatography.Through plasmid site-directed mutagenesis,plasmid restriction digestion,PCR amplification,and homologous recombination,we successfully constructed the recombinant plasmid of EML3 fusion protein containing N-or C-terminal fusion with H3K4M peptide.Sequencing confirmed the correct sequence,and the recombinant plasmid successfully induced the expression of the fusion protein in Escherichia coli.The C-terminal fusion protein(EML3-H3K4M) was cleaved by thrombin,resulting in the release of free H3K4M peptide as well as the EML3 Agenet domain protein.We successfully constructed the recombinant plasmid of EML3 fused with histone H3K4M peptide and obtained a highly pure EML3 fusion protein by IPTG induction and protein purification.
作者
王卓文
刘艳丽
WANG Zhuowen;LIU Yanli(College of Pharmaceutical Science,Soochow University,Suzhou,Jiangsu 215000,China)
出处
《中国科技论文在线精品论文》
2024年第1期79-90,共12页
Highlights of Sciencepaper Online
基金
国家自然科学基金(32271309)。
