MutL融合蛋白的高效表达及其伴侣功能研究(英文)

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 His6 Linkerpeptide 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 .coliK 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 linkerpeptide mutL .重组菌株E .coliAD4 94 (DE3) pET32a linkerpeptide 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 (Ni2 +)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 Linkerpeptide 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

High Expression of Gene Encoding for MutL Fusion Protein and Research on Its Chaperon Function

A mutL gene for DNA mismatch repair (1848 bp) was obtained by PCR amplification from E.coli K 12 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.