High Expression of Gene Encoding for MutL Fusion Protein and Research on Its Chaperon Function
MutL融合蛋白的高效表达及其伴侣功能研究
查看参考文献27篇
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文摘
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A mutL gene for DNA mismatch repair (1848 bp) was obtained by PCR amplification from E. coli K42 genome. The expression vector of the fusion protein Trx-His_6-Linker peptide-MutL ( THLL) was constructed by attaching linker peptide coding sequence and mutL gene to pET32a ( + ) , sequentially. The fusion protein THLL was expressed in E. coli AD494(DE3) by inducing with IPTG. SDS-PAGE revealed that the expected protein with a molecular weight 84 kD was soluble and amounted to about 30% of the total bacterial protein. The fusion protein THLL was purified directly by immobilized metal (Co ~2+ ) chelation affinity chromatography and the purity is over 90 % . Characterization of the fusion protein THLL was performed through non-denaturing gel electrophoresis. The results indicated that the fusion protein THLL could stimulate the binding of the fusion protein Trx-His_6-Linker peptide-MutS (THLS) to DNA but was significantly affected by ATP concentration. |
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其他语种文摘
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DNA错配修复蛋白MutL和其它的修复蛋白相互作用来共同完成大肠杆菌甲基介导的错配修复过程．为研究修复蛋白MutL的体外生物功能构建了融合蛋白Trx-His_6-Linker peotide-MutL(THLL)的表达载体并使其高效表达及易于纯化．mutL基因片段是以E．coli K-12基因组为模板经 PCR扩增获得，并通过基因的体外拼接成功构建了融合蛋白THLL表达载体pET32a-linker peptide- mutL．重组菌株E．coli AD494(DE3)／pET32a-linker peptide-mutL经过IPTG的诱导表达了融合蛋白 TH．LL．收集茵体细胞、超声波破碎后离心取上清进行SDS-PAGE分析，结果表明有一与预期分子量(84 kD)相应的诱导表达条带出现，其表达量约占全细胞蛋白的30％且以可溶形式存在．利用固定化金属离子(Ni~(2+))配体亲和层析柱纯化融合蛋白THLL,其纯度达到90%.通过非变性凝胶电泳分析,对融合蛋白THLL在DNA错配修复过程中的分子伴侣生物功能进行了系统研究.结果表明,THLL能增加融合蛋白Trx-His_6-Linker peptide-MutS(THLS)与含有错配碱基DNA双链的结合,但受ATP的浓度变化影响很大. |
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来源
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中国生物化学与分子生物学报
,2004,20(2):149-155 【核心库】
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关键词
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DNA mismatch repair
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地址
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1.
Wuhan Institute of Virology,Chinese Academy of Sciences, Wuhan, 430071
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Shenyang Institute of Applied Ecology,Chinese Academy of Sciences, Shenyang, 110015
3.
Department of Biochemistry,Imperial College of Science,Technology and Medicine, London
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语种
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英文 |
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文献类型
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研究性论文 |
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ISSN
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1007-7626 |
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学科
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分子生物学 |
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基金
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国家自然科学基金
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国家科技攻关计划项目
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中国科学院重大项目
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文献收藏号
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CSCD:1625435
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