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Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system
Preparative Biochemistry & Biotechnology ( IF 2.9 ) Pub Date : 2022-07-06 , DOI: 10.1080/10826068.2022.2095573
Eva Agustriana 1 , Isa Nuryana 1 , Fina Amreta Laksmi 1 , Kartika Sari Dewi 2 , Hans Wijaya 1 , Nanik Rahmani 1 , Danu Risqi Yudiargo 3 , Astadewi Ismadara 4 , Helbert 5 , Moch Irfan Hadi 6 , Awan Purnawan 1 , Apridah Cameliawati Djohan 1
Affiliation  

Abstract

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.



中文翻译:

嗜热脂肪土芽孢杆菌大片段 DNA 聚合酶 I 在大肠杆菌表达系统中的优化表达

摘要

Bst DNA 聚合酶是一种源自嗜热脂肪土芽孢杆菌的 DNA 聚合酶,具有链置换活性,用于环介导等温扩增 (LAMP) 以快速检测 COVID-19。尽管它有可能用于检测 COVID-19,但使用市售的酶在经济上并不可行。常规使用非商业酶是可取的。然而,印度尼西亚对Bst DNA 聚合酶的研究还很有限。出于这些原因,对重组Bst聚合酶的放大生产进行了初步研究。因此,进行了表达条件的优化。Bst的最佳条件聚合酶表达如下:1 mM IPTG,诱导后孵育时间 6 小时,在 OD 600 1.1 下诱导。与初始条件相比,采用最佳条件可使蛋白质产量增加 2.8 倍。随后,在实验室规模的生物反应器中进行了 1 L 工作体积的操作,然后进行了纯化和透析。1 L 实验室规模生物反应器的最佳结果是通过应用 100 rpm 和 3 vvm 实现的,蛋白质产量为 11.7 mg/L。Bst聚合酶被成功纯化,显示出 813.56 U/mg 的聚合酶活性。

更新日期:2022-07-06
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