Abstract：
Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.
摘要：
核糖核酸酶抑制剂（RIs）是检测和操作RNA必不可少的生物技术工具。如今,由于COVID-19的爆发，高度敏感的RNA检测变得比以往任何时候都更加重要。尽管RNA酶抑制剂的重组表达在大肠杆菌中是可能的,通过各种表达标签维持氧化还原电位和溶解度,使稳健表达变得复杂。在本文中，我们描述了利用改良的转移载体结合ProfinityExactTag和pONE系统的有益特性，在杆状病毒感染的HighFive细胞中大规模表达RI。重组RI以融合形式高水平表达，在柱色谱期间容易裂解。使用随后的阴离子交换色谱作为精制步骤,得到每升培养物12mg天然RI。基于温度依赖性RNase抑制研究,在昆虫细胞中表达的RI显示比市售RI产品(主要在大肠杆菌中产生)更高的热稳定性。不含内毒素的RI变体也可作为安全添加剂应用于未来的治疗剂中以增加基于mRNA的疫苗中的mRNA稳定性。
