火球菌Pyrococcus furious瓣状核酸内切酶1的表达纯化及酶学特征

摘要

[Objective] To clone,express,purify and characterize flap endonuclease 1 from thermophilic archaea Pyrococcusfurious.[Methods] We clonedfen1 gene from Pyrococcusfurious (pFEN1),expressed it in Escherichia coli and purified the protein by affinity chromatography.We applied denaturing polyacrylamide gel electrophoresis to detect the enzymatic activities and studied its interaction with other proteins by using fluorescence labeled oligonucleotides as substrates.[Results] pFEN1 was overexpressed in E.coli.The presence of salts diminished the endonuclease activity of pFEN 1,with cleavage greatly reduced at 100 mmol/L of NaCl.The activity of pFEN 1 was detected only in the presence of magnesium (Mg2+) or manganese (Mn2+),and pFEN1 showed a higher endonucease activity under the catalysis of Mn2+.pFEN1 was thermally stable and had highest activity at temperature range of 60-65 ℃C.Proliferating cell nuclear antigen (PCNA) can significantly promote the activity of pFEN1.[Conclusion] This study confirmed that pFEN1 is a Mg2+ or Mn2+ dependent endonuclease and PCNA can stimulate its activity.%[目的]克隆表达和纯化火球菌Pyrococcus furious来源的瓣状核酸内切酶1基因pFEN1 (PF1414),对该蛋白的活性和酶学特征进行鉴定和分析.[方法]将pFEN1在大肠杆菌中进行重组表达,经亲和层析纯化得到电泳纯蛋白;利用人工合成的荧光标记的寡核苷酸片段作为底物,用变性聚丙烯酰胺凝胶电泳鉴定pFEN1在体外的酶学特性以及与其他蛋白的相互作用.[结果]pFEN1重组蛋白能在大肠杆菌中进行高效表达;高于100 mmol/L的NaC1会抑制pFEN1的活性;pFEN1的核酸酶活性依赖于金属离子Mg2+或Mn2+,且Mn2+的催化效率优于Mg2+;来自嗜热古菌的pFEN1是一种耐高温蛋白,最适反应温度为60-65℃C;增殖细胞核抗原(PCNA)能促进pFEN1的内切酶活性.[结论]本研究证实pFEN1是一种Mg2+或Mn2+依赖的核酸内切酶,且PCNA能促进该酶的活性.