生命科学研究2018，Vol.22Issue(4)：291-297,304,8.DOI:10.16605/j.cnki.1007-7847.2018.04.005
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
摘要
Abstract
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.关键词
聚合酶链式反应(PCR)/DNA聚合酶/Pfu酶/蛋白质纯化/Ni-NTA/制备方法Key words
polymerase chain reaction (PCR)/DNA polymerase/Pfu polymerase/protein purification/Ni-NTA/preparation method分类
生物科学引用本文复制引用
韦慧,曹贤明,李昱龙,涂中华,樊奔..Pfu DNA聚合酶的制备过程及其条件优化[J].生命科学研究,2018,22(4):291-297,304,8.基金项目
国家自然科学基金青年科学基金项目(31100081) （31100081）
江苏省自然科学基金资助项目(BK20151514) （BK20151514）
江苏省高校自然科学研究重大项目(17KJA220001) （17KJA220001）
句容市农业科技支撑计划(NY2017095974) （NY2017095974）
