嗜热古菌Archaeoglobus fulgidus RecJ核酸酶的表达纯化及酶学特征

[目的]克隆表达嗜热古菌Archaeoglobus fulgidus(A.fulgidus)来源的RecJ核酸酶基因(ORF编号AF_0699,NCBI数据库基因登陆号为AF_RS03550),对该重组蛋白的核酸酶活性及酶学特征进行鉴定和分析.[方法]将A.fulgidus RecJ(AfuRecJ)核酸酶在大肠杆菌中...

Expression, purification and characterization of RecJ nuclease from Archaeoglobus fulgidus

[Objective] To clone, express and purify the RecJ nuclease gene(AF_0699) from Archaeoglobus fulgidus, identify and analyze its nuclease activity. [Methods] Recombinant RecJ nuclease(AfuRecJ) was expressed in E. coli and purified by affinity chromatography. The enzymatic properties of AfuRecJ nuclease were characterized in vitro using synthesized oligo(deoxy) nucleotides as substrate. [Results] AfuRecJ nuclease has a single-stranded DNA specific 3’–5’ exonuclease activity, which depends on the divalent metal ions Mn2+ or Mg2+, and the catalytic efficiency of Mn2+ is significantly higher than that of Mg2+. The optimal reaction temperature is between 55 and 65 ℃. The presence of high concentration of NaCl decreases the exonuclease activity of AfuRecJ, and cleavage percentage greatly reduces at 200 mmol/L of NaCl. AfuRecJ shows 3’–5’ exonuclease activity on single-stranded RNA, higher than that of single-stranded DNA. The 3’ terminal phosphate group inhibits the AfuRecJ nuclease. AfuRecJ has different preferences for substrate length. It can effectively hydrolyze ssRNA≥4 nt, and ssDNA≥12 nt. Moreover, the hydrolysis activity of AfuRecJ on dsDNA with special structure(3’ overhang and 3’ fork) is similar to that of ssDNA. [Conclusion] The nuclease activity of AfuRecJ depends on the Mn2 + and can cleave ssDNA and ssRNA with a preference for RNA substrate.