Pfu DNA聚合酶的制备过程及其条件优化

Preparation of Pfu DNA Polymerase with Optimized Purification Procedures

聚合酶链式反应(polymerase chain reaction,PCR)可以方便、快捷、准确地大量复制目的基因片段,是分子生物学研究中不可或缺的技术手段。在PCR反应中,DNA聚合酶起着关键作用。Pfu酶(Pfu DNA polymerase)是DNA聚合酶的一种,具有5′→3′聚合酶活性和3′→5′外切核酸酶活性,可在聚合酶链式反应中纠正错误掺入的碱基,可以快速、高保真地扩增DNA片段,在分子克隆和DNA测序当中运用十分广泛。本研究建立了一套Pfu聚合酶表达与纯化的详尽方案,该方案利用异丙基硫代-β-D-半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)作为诱导剂,在大肠杆菌BL21(DE3)中异源表达Pfu酶,然后利用Ni-NTA柱层析纯化制备。研究中对IPTG诱导时间、细胞破碎条件、缓冲液类型及其p H、粗酶液热处理温度与时间、洗脱液中最佳咪唑浓度等重要条件和参数分别进行了优化确定。实验结果表明,使用该方案可以成功制备高产量高纯度的Pfu聚合酶,完全满足常规PCR反应的要求。本研究建立的实验方法和技术参数,十分适用于研究者小规模自主制备Pfu酶,对于有大量分子克隆要求的课题组,利用该方案自主制备Pfu酶能够显著节省科研成本。

Polymerase chain reaction（PCR）, an indispensable technical tool in molecular biology research, per-mits the amplification of a large numbers of DNA segments easily and rapidly. In the PCR, DNA polymerase plays a key role. Pfu DNA polymerase is a kind of DNA polymerase with 5′→3′ polymerase activity and 3′→5′ exonuclease activity, which can correct the erroneously incorporated bases in the polymerization reaction,therefore Pfu is able to amplify DNA fragments with high fidelity and is widely used in molecular cloning and DNA sequencing. Herein, a set of detailed protocol for the expression and purification of Pfu polymerase was established. In this protocol isopropyl β-D-thiogalactoside（IPTG） was used as an inducer for the heterologous expression of Pfu polymerase in E. coli BL21（DE3）; then purification was performed using Ni-NTA column chromatography. A series of important conditions and parameters were optimized and determined, includ-ing IPTG-inducing time, buffer type and p H of bacterial cell disruption, time and temperature of heating treatment of crude proteins, optimum imidazole concentration of eluent. The experimental results showed that Pfu polymerase can be prepared in high yield and purity with this protocol. The obtained Pfu fully meets the requirements of PCR reaction. The experimental conditions and the technical parameters established in this study are suitable for production of Pfu in many labs in a small scale. The self-made Pfu could significantly reduce the cost of experiments in which a large amount of molecular cloning work is needed.