嗜热细菌Anaerobaculum hydrogeniforman Pif1解旋酶的异源表达与解旋反应特性

Heterologous expression and characterization of unwinding reaction of Pif1 helicase from Anaerobaculum hydrogeniforman

摘要:

[目的]原核表达与纯化源自嗜热细菌Anaerobaculum hydrogeniforman的Pif1解旋酶，从而探究其解旋反应特性。[方法]将重组载体pET21a-AnaPif1-TEV/SUMO导入大肠杆菌BL21（DE3）中诱导表达，并进行Ni-NTA亲和层析、Superdex200凝胶过滤层析等一系列纯化手段；再利用快速停留监测技术系统地研究Ana.Pif1的解旋反应特性，包括解旋极性、最佳ATP浓度、最佳金属辅因子、最佳解旋温度以及解旋复制中间体DNA的底物特异性。[结果]通过异源表达与纯化，获得了无任何标签序列、分子量59 kDa的Ana.Pif1蛋白，纯度达97%，产率为9.5 mg/L。本研究首先验证Ana.Pif1具有5'-3'的解旋极性，并发现其解旋反应最佳ATP浓度为2 mmol/L，其最佳二价金属辅因子为Mg²⁺，最适反应温度为55℃。解旋复制中间体的底物特异性显示，Y-S型复制叉的解旋速率最高，为0.127 s^–1；而12 nt-bubble底物的解旋幅度最大，达到78.8%；暗示这些底物可能是Ana.Pif1的天然底物。[结论]本文首次较为系统地分析了Ana.Pif1解旋酶的解旋反应特性，为阐明此类嗜热细菌Pif1解旋酶的分子作用机制奠定了基础。

Abstract:

[Objective] We expressed and purified a Pif1 helicase from Anaerobaculum hydrogeniforman to assess its unwinding characteristics.[Methods] Recombinant vector pET21a-Ana.Pif1-TEV/SUMO was constructed and expressed in E. coli BL21(DE3), and Ana.Pif1 protein was purified using Ni-NTAs and Superdex 200 column chromatography. We then evaluated the unwinding polarity, optimal ATP concentration, optimal metal cofactor, optimal unwinding temperature, and substrate specificity of Ana.Pif1 by stopped-flow FRET assay. [Results] The molecular weight of Ana.Pif1 helicase was 59 kDa with no tags, protein yield was 9.5 mg/L, and purity was about 97%. We confirmed the unwinding polarity of Ana.Pif1 was from DNA 5' to 3' end. And we found the optimal ATP concentration for Ana.Pif1 unwinding to be 2 mmol/L, the optimal metal cofactor to be Mg²⁺, and the optimal reaction temperature to be 55℃. The substrate specificity of Ana.Pif1 unwinding DNA replication intermediates indicated that the Y-S fork substrate had the highest unwinding rate, 0.127 s^-1, and 12 nt-bubble substrate had the largest unwinding amplitude, 78.8%, indicating these two substrates could be natural replication intermediates for Ana.Pif1. [Conclusion] This study is the first to systematically analyze the unwinding characteristics of Ana.Pif1 helicase. These findings facilitate elucidation of the molecular mechanisms of these thermophilic bacteria Pif1-family helicases.