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

Expression,purification and characterization of RecJ nuclease from Archaeoglobus fulgidus

【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;利用人工合成的带有末端荧光标记的寡核苷酸作为底物,体外反应后,利用8 mol/L尿素变性聚丙烯酰胺凝胶电泳鉴定AfuRecJ核酸酶的水解产物。【结果】AfuRecJ核酸酶具有单链DNA特异性的3’–5’外切核酸酶活性,酶活性依赖于二价金属离子Mn2+或Mg2+,且Mn2+存在条件下的催化效率明显优于Mg2+;其最适反应温度范围为55–65℃;高于200 mmol/L的NaCl会显著抑制AfuRecJ的核酸酶活性。AfuRecJ还具有单链RNA 3’–5’外切酶活性,且活性高于单链DNA底物。单链核酸3’末端的磷酸基团对水解活性有一定抑制作用。AfuRecJ对单链核酸的长度有一定的选择性,可以有效水解长度≥4个核苷酸长度的单链RNA、≥12个核苷酸长度的单链DNA,而且对双链DNA中的3’单链DNA结构(3’突出单链尾巴与末端分叉结构)具有类似单链DNA的水解活性。【结论】本研究证实AfuRecJ是一种单链核酸特异性的3’–5’外切核酸酶,且相比单链DNA,单链RNA为优势底物,推测其在胞内可能参与RNA降解与DNA修复。

[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.