Overexpressing and Purifying a Toxic Nuclease from <em>Escherichia coli</em>

Escherichia coli has been widely employed as an expression system to produce recombinant proteins. Yet some types of recombinant proteins, such as nucleases, can be difficult to overexpress recombinantly in E. coli due to their enzymatic activity on cellular DNA or RNA. Such toxic effects can lead to slow growth and poor protein yields. One such example is the endoribonuclease Nsp15 from coronaviruses: yields of wild-type (WT) gamma-coronavirus Nsp15 are much lower than catalytically inactive Nsp15. To address this issue, we have employed the use of C41(DE3) cells and same-day single colony starter cultures to help reduce toxicity. Recombinant Nsp15 containing an N-terminal 6xHis-tag was overexpressed and purified using cobalt-based affinity chromatography followed by size exclusion chromatography (SEC). The resulting yields of purified protein are sufficient for biochemical assays and cryo-electron microscopy structural studies. This approach has proven effective in mitigating the toxic effects and achieving satisfactory protein yields for further research analysis.