Abstract
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 µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a Kiapp of 75.80 μg/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.
Graphical abstract
Key points
• Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3).
• The slow-binding inhibition was shown by plotting the apparent first-order rate constant (k obs ).
• Protein–protein interaction between SnuCalCpIs and papain was verified by docking simulation.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Funding
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1A2B5B01002596) and “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01589402)” provided by Rural Development Administration, Republic of Korea.
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CWK and BC conceived and designed research. BC conducted experiments. CWK, BC, and SHY wrote the manuscript draft. PSC edited the manuscript. All authors read and approved the manuscript.
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Kwon, C.W., Chung, B., Yoo, SH. et al. Heterologous expression of a papain-like protease inhibitor (SnuCalCpI17) in the E. coli and its mode of inhibition. Appl Microbiol Biotechnol 106, 4563–4574 (2022). https://doi.org/10.1007/s00253-022-12032-8
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DOI: https://doi.org/10.1007/s00253-022-12032-8