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优化生产可溶性E.E.生产条件生物技术应用中的聚甲基聚合酶

Igor P Oscorbin¹, Maria S Kunova^1,2, Maxim L Filipenko¹

¹Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), 8, Lavrentiev Avenue, Novosibirsk 630090, Russia.

|January 25, 2025

概括

优化Escherichia coli中的重组聚合酶 (PAP 1) 生产对于RNA应用至关重要. BL21 (DE3) pLysS菌株产生了最好的PAP 1蛋白平衡,提高了其生物技术效用.

科学领域:

生物化学生物化学
分子生物学分子生物学
生物技术是生物技术.

背景情况:

来自大肠杆菌的多甲基聚合酶 (PAP 1) 在RNA上合成多甲基尾巴,这对细菌RNA降解至关重要.
在体外,PAP 1对于RNA测序库的准备和mRNA疫苗生产至关重要.
大肠杆菌PAP 1表现出毒性和不稳定性,阻碍其有效表达和净化.

研究的目的:

为了优化Escherichia coli重组聚合酶 (PAP 1) 的生产.
评估不同的大肠杆菌菌株和表达条件,以提高PAP 1的产量,溶解度和活性.

主要方法:

在七种不同的大肠杆菌菌株中表达大肠杆菌PAP 1.
在各种条件下评估蛋白质产量,可溶性和酶活性.
诱导温度和等离子体拷贝数对PAP 1积累的影响的分析.

主要成果:

这种BL21 (DE3) pLysS大肠杆菌菌株表现出细胞密度,PAP 1产量,溶解度和特异性活性的最佳平衡.
罗塞塔2 (DE3) 和罗塞塔蓝 (DE3) 菌株的溶解性较差,可能是由于高的翻译效率.
增加的诱导温度 (>18°C) 对PAP 1的溶解度产生了负面影响.
PAP 1积累与等离子体拷贝数正相关,这表明它可以用作标记物.

结论:

BL21 (DE3) pLysS是用于重组PAP 1生产的首选大肠杆菌菌株.
表达条件,包括诱导温度和菌株选择,显著影响PAP 1的产量和溶解性.
等离子体拷贝数可以作为PAP 1活动和生产水平的替代标记.

关键词:

美国大肠杆菌 (E. coli).PAP 1 1 一个问题这就是为什么pcnBB.聚A) 聚合酶.聚亚脱化聚亚脱化聚亚脱化蛋白质表达蛋白质表达重组蛋白质蛋白质的重组蛋白质是什么?

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