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.
Highlights
Ribonuclease inhibitors are a 50 kDa cysteine and leucine-rich repeat protein found in the cytosol of cells in most mammalian species
We focused on the expression of Ribonuclease inhibitors (RIs) in the Baculovirus Expression Vector System (BEVS) system where the chance of correct folding and producing more stable proteins is significantly higher than in E. coli
Recombinant RI was expressed in baculovirus/insect cell expression system [15], which is based on the co-transfection of insect cells with linearized Autographa californica multiple-capsid nuclear polyhedrosis virus (AcMNPV) baculovirus DNA and a transfer plasmid carrying the gene of interest, for the construction of baculovirus vectors
Summary
Ribonuclease inhibitors are a 50 kDa cysteine and leucine-rich repeat protein found in the cytosol of cells in most mammalian species. After initial oxidation of a small number of cysteine residues, a conformational change occurs, resulting in the cooperative formation of 15 disulfide bonds that leads to the inactivation of the porcine RI. RI was originally purified from placenta [10], while the first recombinant RI (porcine) in an active form was expressed in S. cerevisiae [6], at a low yield (0.2 mg recombinant protein/g wet cells). Addition of the reducing agent dithiotreitol (DTT), low production temperature and co-expression of the chaperonin GroELS resulted in high level production using the conditions with the EnBase® technology [5,12]. Another promising attempt was to use N-terminal tags to increase the solubility of RI. We focused on the expression of RI in the Baculovirus Expression Vector System (BEVS) system where the chance of correct folding and producing more stable proteins is significantly higher than in E. coli
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