Abstract:Polymerase chain reaction (PCR), an indispensable technical tool in molecular biology research, permits 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 col-umn chromatography. A series of important conditions and parameters were optimized and determined, including IPTG-inducing time, buffer type and pH 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.
引用本文:
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.
WEI Hui, CAO Xian-ming, LI Yu-long, TU Zhong-hua, FAN Ben. Preparation of Pfu DNA Polymerase with Optimized Purification Procedures. Life Science Research, 2018, 22(4): 291-297.