Heterologous expression of a papain-like protease inhibitor (SnuCalCpl17) in the E. coli and its mode of inhibition

The effect of the Escherichia coli (E. coli) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the E. coli BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in E. coli Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC50) of 105.671 +/- 9.857 mu g/mL and a slow-binding inhibition mode against papain at pH 7.0 with a K-i(app) of 75.80 mu g/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC50 of the inhibitor.

Automated summary

This study evaluated the expression of recombinant papain-like cysteine protease inhibitors in the Escherichia coli Rosetta (DE3) system, and determined the inhibition mode of the expressed inhibitors. SnuCalCpI08 and SnuCalCpI17 were successfully expressed in this system, with SnuCalCpI17 showing a slow-binding inhibition mode in papain inhibition assay.