水热
热启动PCR
聚合酶
多重位移放大
DNA聚合酶
聚合酶
分子生物学
变性(裂变材料)
聚合酶链反应优化
生物
DNA
聚合酶链反应
化学
DNA提取
生物化学
套式聚合酶链反应
基因
核化学
作者
Nosaibah Samman,Khawlah Al-Muhalhil,Atef Nehdi
标识
DOI:10.1016/j.jksus.2023.102565
摘要
Taq DNA polymerase from Thermus aquaticus (Taq) is a widely used enzyme in molecular biology research and clinical diagnostics because of its utility in DNA amplification and sequencing assays. For laboratory large-scale production of enzymes, effort, time and cost are crucial factors. Here we describe a simple and efficient method for the production of high-quality recombinant Taq DNA polymerase using a combined method based on heat denaturation and nickel affinity chromatography. His-tagged Taq DNA polymerase was overexpressed in Escherichia coli and isolated in a first step by heat denaturation of the bacterial lysate, since Taq DNA polymerase is a thermo-resistant protein, it remains in the soluble fraction with a few bacterial protein contaminants. The isolated His-tagged Taq DNA polymerase was further purified by nickel-NTA chromatography leading to a high quality and purity enzyme with a comparable activity to most of commercially available counterparts. DNA-contaminated Taq DNA polymerases constitute a major concern especially during DNA amplification because they can lead to generation of non-specific products. Since Taq DNA polymerase is a DNA binding protein, it carries during purification process bacterial DNA contaminants. We treated the purified Taq DNA polymerase with DNase to end up with a high-quality DNA-free Taq DNA polymerase.
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