Codon optimization of a gene encoding DNA polymerase from Pyrococcus furiosus and its expression in Escherichia coli.

IF 3.6 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal, genetic engineering & biotechnology Pub Date : 2023-11-21 DOI:10.1186/s43141-023-00605-7
Isa Nuryana, Fina Amreta Laksmi, Kartika Sari Dewi, Faiz Raihan Akbar, Nurhayati, Rikno Harmoko
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Abstract

Background: DNA polymerase is an essential component in PCR assay for DNA synthesis. Improving DNA polymerase with characteristics indispensable for a powerful assay is crucial because it can be used in wide-range applications. Derived from Pyrococcus furiosus, Pfu DNA polymerase (Pfu pol) is one of the excellent polymerases due to its high fidelity. Therefore, we aimed to develop Pfu pol from a synthetic gene with codon optimization to increase its protein yield in Escherichia coli.

Results: Recombinant Pfu pol was successfully expressed and purified with a two-step purification process using nickel affinity chromatography, followed by anion exchange chromatography. Subsequently, the purified Pfu pol was confirmed by Western blot analysis, resulting in a molecular weight of approximately 90 kDa. In the final purification process, we successfully obtained a large amount of purified enzyme (26.8 mg/L). Furthermore, the purified Pfu pol showed its functionality and efficiency when tested for DNA amplification using the standard PCR.

Conclusions: Overall, a high-level expression of recombinant Pfu pol was achieved by employing our approach in the present study. In the future, our findings will be useful for studies on synthesizing recombinant DNA polymerase in E. coli expression system.

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炽热焦球菌DNA聚合酶基因密码子优化及其在大肠杆菌中的表达。
背景:DNA聚合酶是DNA合成PCR检测的重要组成部分。改进DNA聚合酶,使其具有强大的分析所必需的特征,这是至关重要的,因为它可以广泛应用。Pfu DNA聚合酶(Pfu pol)是一种高保真度的聚合酶,源自于狂热焦球菌(Pyrococcus furiosus)。因此,我们的目标是利用密码子优化的合成基因开发Pfu pol,以提高其在大肠杆菌中的蛋白产量。结果:通过镍亲和层析和阴离子交换层析两步纯化,成功表达并纯化了重组Pfu pol。随后,纯化的Pfu pol经Western blot分析证实,分子量约为90 kDa。在最后的纯化过程中,我们成功获得了大量的纯化酶(26.8 mg/L)。此外,纯化的Pfu pol在使用标准PCR进行DNA扩增时显示出其功能和效率。结论:总的来说,采用我们的方法在本研究中实现了重组Pfu pol的高水平表达。本研究结果将为在大肠杆菌表达体系中合成重组DNA聚合酶的研究提供参考。
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