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.
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| http://dx.doi.org/10.3390/biom12020273 | DOI Listing |
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