Modifying a bacterial tyrosinase zymogen for use in protease activity assays
Modifying a bacterial tyrosinase zymogen for use in protease activity assays
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修饰细菌酪氨酸酶酶原用于蛋白酶活性测定
DOI：
10.1007/s00253-022-12284-4
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发表时间：
2022-11
期刊：
影响因子：
--
通讯作者：
Lihui Zou
中科院分区：
文献类型：
--
作者：
Junhua Zhang;Wei Huang;Lanxin Zhang;Xiaokun Tang;Gaoyuan Sun;Lihui Zou
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 fromVerrucomicrobium 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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