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

摘要:

[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。

Abstract:

[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.