火球菌8-氧鸟嘌呤DNA糖苷酶基因克隆、表达纯化和酶学性质研究

Cloning expression and biochemical characterizations of 8-oxoguanine DNA glycosylase from Pyrococcus furiosus

摘要:

【目的】在大肠杆菌中表达火球菌8-氧鸟嘌呤DNA糖苷酶，纯化得到重组火球菌8-氧鸟嘌呤DNA糖苷酶，在此基础上系统研究火球菌8-氧鸟嘌呤DNA糖苷酶的酶学特征。【方法】构建8-氧鸟嘌呤DNA糖苷酶重组表达质粒，将重组质粒转化Escherichia coli Rosetta (DE3)，利用IPTG诱导表达重组蛋白，通过Ni2+亲和层析柱纯化重组蛋白；最后利用含8-氧鸟嘌呤损伤的寡核苷酸作为底物，测定8-氧鸟嘌呤DNA糖苷酶的酶学性质。【结果】在大肠杆菌中成功诱导表达了重组火球菌8-氧鸟嘌呤DNA糖苷酶，经Ni2+亲和纯化后蛋白纯度大于95%。在体外鉴定了重组火球菌8-氧鸟嘌呤DNA糖苷酶的酶学性质。结果表明重组火球菌8-氧鸟嘌呤DNA糖苷酶可以切除DNA中的8-氧鸟嘌呤(8-Oxo-G，GO)损伤碱基，并且具有AP裂解酶活性。重组火球菌8-氧鸟嘌呤DNA糖苷酶催化反应的最适pH值和温度分别是pH 8.5和55 °C。除Zn2+对火球菌8-氧鸟嘌呤DNA糖苷酶的酶促反应有明显的抑制作用外，实验中测定的其它二价离子(Mn2+，Mg2+，Ca2+，Ni2+，Co2+，Cu2+)对其没有明显的影响。离子强度在50?100 mmol/L范围内对其酶促反应影响不大，超过100 mmol/L时有明显的抑制作用。与8-氧鸟嘌呤互补的碱基差异对火球菌8-氧鸟嘌呤DNA糖苷酶切除8-氧鸟嘌呤损伤的效率影响不大；但与单链DNA相比，双链DNA是优选底物，切割效率如下：GO/C≈GO/G≈GO/T≈GO/A>GO/?。【结论】在大肠杆菌中成功表达，并Ni2+亲和纯化了火球菌8-氧鸟嘌呤DNA糖苷酶，生化研究表明制备的重组蛋白具有8-氧鸟嘌呤DNA糖苷酶活性，可能负责切除火球菌基因组DNA中的8-氧鸟嘌呤损伤。

Abstract:

[Objective] Express and purify the Pyrococcus furiosus (P. furiosus) 8-oxoguanine DNA glycosylase; characterize the enzymatic properties of the recombinant P. furiosus 8-oxoguanine DNA glycosylase. [Methods] First, pfogg gene was cloned into an expression vector. Second, the expression of the recombinant protein was induced in Escherichia coli Rosetta (DE3) by IPTG. Third, the recombinant protein was purified through Ni2+ affinity chromatography. Finally, the enzymatic reaction of PfOgg was biochemically characterized using different oligonucleotides as substrates. [Results] The recombinant P. furiosus 8-oxoguanine DNA glycosylase was successfully expressed in E. coli and the purity was up to 95%. The enzymatic characterizations of recombinant P. furiosus 8-oxoguanine DNA glycosylase were assayed in vitro. The results showed that (1) the recombinant P. furiosus 8-oxoguanine DNA glycosylase could efficiently remove the 8-oxoguanine damage from both single-stranded and double-stranded DNA and the excision efficiency of 8-oxoguanine from various DNA substrates was the following order: GO/C≈GO/G≈GO/T≈GO/A>GO/?; (2) the optimal pH value and temperature for the reaction were pH 8.5 and 55 °C, respectively; (3) Zn2+ clearly inhibited the removal of 8-oxo-G by PfOgg, and the other divalent ions, such as Mn2+, Mg2+, Ca2+, Ni2+, Co2+ and Cu2+ had no significant effect on the enzymatic reaction; (4) the removal of 8-oxo-G was highly efficient with the ionic strength ranging from 50 to 100 mmol/L, but was clearly inhibited by higher ionic strength (above 100 mmol/L). (5) the PfOgg is highly heat-resistant, with a half lifetime of 5 min at 100 °C. [Conclusion] P. furiosus 8-oxoguanine DNA glycosylase was successfully expressed in E. coli, and had the typical enzymatic activities of removing the 8-oxoguanine damaged base from single-stranded and double-stranded DNA.