核酸外切酶Ⅷ截短体的重组表达及其在体外DNA重组反应中的应用

Recombinant expression of truncated exonuclease Ⅷ and its application in in vitro DNA recombination

摘要:

核酸外切酶Ⅷ (Exonuclease Ⅷ，Exo Ⅷ) 是一种不依赖于ATP的dsDNA 5?-3?核酸外切酶，可作为体外DNA重组反应极具应用价值的候选蛋白。目前关于Exo Ⅷ在体外DNA重组反应中的应用尚未有文献报道。本研究构建了保留完整外切活性的截短Exo Ⅷ (Truncated exonuclease Ⅷ，tExo Ⅷ) 的重组表达载体pET28a-tExo Ⅷ，实现了tExo Ⅷ在大肠杆菌中的高效表达，在纯化获得高纯度蛋白的基础上，对体外重组反应的温度、反应时间、同源臂长度等因素进行了优化分析。研究结果表明，tExo Ⅷ在大肠杆菌中以可溶性形式高效表达，每升可纯化92.40 mg tExo Ⅷ，比活力为1.21×105 U/mg；在10 μL的重组体系中，2.5 U的tExo Ⅷ于25 ℃反应12.5 min随后50 ℃保温50 min时重组效率最高。添加Pfu DNA聚合酶的体外同源臂延伸策略可以有效提高重组克隆的效率。以转化效率为2.2×106 CFU/μg的Mach1T1为受体细胞，对于含有21 bp同源臂的1 kb片段与5.8 kb线性化载体的重组，每微克载体可形成1.1×104个重组克隆，且阳性率大于80%。同源臂长度在8–21 bp范围内，重组反应效率随着同源臂长度增加而提高。在最佳反应条件下，同源臂长度仅为8 bp仍可实现有效的重组反应。Exo Ⅷ介导的体外重组体系具有酶制备方法简单、对DNA的克隆无酶切位点限制及高重组克隆效率等显著优点，是分子生物学领域具有潜在应用价值的高效基因克隆新体系。

Abstract:

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5′-3′ exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×105 U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×106 cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×104 recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.