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Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Preparative Biochemistry & Biotechnology ( IF 2.9 ) Pub Date : 2022-10-28 , DOI: 10.1080/10826068.2022.2137731
Kashif Maseh 1 , Syed Farhat Ali 1 , Shazeel Ahmad 2 , Naeem Rashid 2
Affiliation  

Abstract

Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 °C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage conditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing overnight at −20 °C and thawing for 45 minutes at 28 °C). Purified Pca-Pol possessed 3′–5′ exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity.



中文翻译:

经济高效、高产地生产用于 PCR 应用的 Pyrobaculum calidifontis DNA 聚合酶

摘要

聚合酶链反应(PCR)广泛用于克隆、基因工程、诱变、检测和诊断。PCR 需要热稳定性 DNA 聚合酶。在这里,我们描述了Pyrobaculum calidifontis DNA 聚合酶 ( Pca -Pol)的低成本和高回收率生产。该基因被克隆到pET-28a中并在大肠杆菌BL21CodonPlus中表达。优化了基因表达条件。最终,用 0.1 mM IPTG 在 37 °C 下诱导基因表达 3 小时。产生的重组Pca -Pol 通过固定金属离子亲和层析纯化至均质,每升培养物产生约 9000 U 的Pca -Pol,回收率为 92%。稳定性和PCR扩增效率Pca -Pol 在各种储存条件下进行了测试,在含有 0.1% Tween 20、0.2 mg/mL BSA 和 20% 甘油的 25 mM Tris-Cl 缓冲液 (pH 8.5) 中效率最高。在此条件下,即使储存一年后,也未观察到Pca -Pol的 PCR 活性损失。然而,反复冻融会降低Pca -Pol 的酶活性。7 次延长冻融循环后保留 55% PCR 扩增活性(-20 °C 冷冻过夜,28 °C 解冻 45 分钟)。纯化的Pca -Pol 具有 3'–5' 核酸外切酶(校对)活性,与不具有校对活性的Taq聚合酶相比,预计具有更高的保真度。

更新日期:2022-10-28
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