Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells

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.

Keywords: baculovirus-insect expression system; high-level soluble expression; quantitative PCR; ribonuclease inhibitor.

Figure 1

Purified RI run on SDS-PAGE.

Figure 2

Remaining activity of RI after 30 min incubation at elevated temperatures. In each block, pre-heat temperatures are indicated. RNA is run on an e-gel after incubation with RNase A and the pre-heated RI. In all measurements, lane 1 denotes ‘insect RI’; lane 2 ‘RI I’, lane 3 ‘RI II’; lane 4 ‘RI III’; and lane 5 ‘RI IV’. RNA denotes the experiments without adding either RNase nor RI, and lane RNase is with added RNase, but no RI. Panel A: Remaining activity of RIs with no pre-incubation. Panel B: Remaining activity of RIs with pre-incubation at 60 °C and 62.5 °C on the left and right side respectively Panel C: Remaining activity of RIs with pre-incubation at 65 °C and 67.5 °C on the left and right side respectively.

Figure 3

Melting profile of each ribonuclease inhibitor alone and in the presence of excess RNase A. Gree-Insect RI; Orange-‘RI I’; Blue-‘RI II’; Turquoise-‘RI III’; Pink-‘RI IV’. On every panel, the lighter colored curves correspond to free RI, while the darker for the RI-RNase A complex.