TargetEx Ltd., Madách I. u. 31/2., H-2120 Dunakeszi, Hungary.
Biomolecules. 2022 Feb 8;12(2):273. doi: 10.3390/biom12020273.
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 , 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 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 ) 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.
核糖核酸酶抑制剂（RIs）是检测和操作 RNA 不可或缺的生物技术工具。如今，由于 COVID-19 的爆发，对 RNA 的高灵敏度检测变得比以往任何时候都更加重要。尽管可以在 中重组表达 RNase 抑制剂，但通过各种表达标签维持氧化还原电势和溶解度会使稳健表达变得复杂。在本文中，我们描述了利用结合了 Profinity Exact Tag 和 pONE 系统的有益特性的改良转移载体，在杆状病毒感染的 细胞中大规模表达 RI。重组 RI 以融合形式高表达，在柱上色谱过程中很容易被切割。随后进行阴离子交换色谱作为抛光步骤，每升培养物可产生 12 毫克天然 RI。基于温度依赖性 RNase 抑制研究，昆虫细胞中表达的 RI 比市售的 RI 产品（主要在 生产）具有更高的热稳定性。无内毒素的 RI 变体也可应用于未来的治疗学中，作为一种安全的添加剂，增加 mRNA 疫苗中 mRNA 的稳定性。
