Alternative TitleHigh Expression of Gene Encoding for MutL Fusion Protein and Research on Its Chaperon Function
Bi Lijun1; Zhou Yafeng1; Zhang Xianen1; Zhang Zhiping1; Zhang Chenggang2; Anthony E G CASS3
Source Publication中国生物化学与分子生物学报
AbstractDNA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-His6-Linker peptide-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.coil 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 peptidemutL.重组菌株E.coli AD94(DE3)/pET32a-linker peptide-mutL经过IPTG的诱导表达了融合蛋白THLL.收集菌体细胞、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(84kD)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-His6-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.
Other AbstractA 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.
KeywordMutL融合蛋白 表达 基因 PCR 连接肽
Indexed ByCSCD
Citation statistics
Cited Times:1[CSCD]   [CSCD Record]
Document Type期刊论文
Affiliation1.Wuhan Institute of Virology,Chinese Academy of Sciences
3.Department of Biochemistry,Imperial College of Science,Technology and Medicine
Recommended Citation
GB/T 7714
Bi Lijun,Zhou Yafeng,Zhang Xianen,等. MutL融合蛋白的高效表达及其伴侣功能研究[J]. 中国生物化学与分子生物学报,2004,20.0(002):149-155.
APA Bi Lijun,Zhou Yafeng,Zhang Xianen,Zhang Zhiping,Zhang Chenggang,&Anthony E G CASS.(2004).MutL融合蛋白的高效表达及其伴侣功能研究.中国生物化学与分子生物学报,20.0(002),149-155.
MLA Bi Lijun,et al."MutL融合蛋白的高效表达及其伴侣功能研究".中国生物化学与分子生物学报 20.0.002(2004):149-155.
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