Abstract
Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients.
Key points
• RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase.
• N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase.
• The activity of caspase-3 could be measured by the zymogen activation system.
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Data availability
The datasets generated and/or analyzed during this study are included in this manuscript (and its supplementary information file). Any additional data, if needed, will be available with the corresponding author upon reasonable request.
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Acknowledgements
We are grateful to Professor Fei Xiao for his valuable suggestions on the experimental design. We are grateful to Professor Meng Wang for his valuable suggestions on the enzymatic activity analysis.
Funding
This study was funded by Beijing Hospital Project BJ-2020–134 and the National Natural Science Foundation of China (81871107).
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JZ and LZ conceived and designed research. JZ, WH, LZ, and XT conducted experiments. JZ and GS analyzed data. JZ and LZ wrote the manuscript. All authors read and approved the manuscript.
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Zhang, J., Huang, W., Zhang, L. et al. Modifying a bacterial tyrosinase zymogen for use in protease activity assays. Appl Microbiol Biotechnol 106, 8285–8294 (2022). https://doi.org/10.1007/s00253-022-12284-4
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DOI: https://doi.org/10.1007/s00253-022-12284-4