Establishment of in vitro biological activity evaluation system for OTULIN
10.12206/j.issn.2097-2024.202601035
- VernacularTitle:去线性泛素化酶体外生物活性评价体系的构建
- Author:
Zhewei XIA
1
;
Yu LI
1
;
Chunquan SHENG
1
Author Information
1. School of Pharmacy, Naval Medical University, Shanghai 200433, China.
- Publication Type:Originalarticles
- Keywords:
OTULIN;
linear ubiquitin;
in vitro bioactivity;
enzyme activity evaluation
- From:
Journal of Pharmaceutical Practice and Service
2026;44(5):259-263
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish an in vitro enzymatic activity evaluation system for OTULIN （OTU domain DUB with linear linkage specificity）and provide an experimental basis for OTULIN inhibitor screening. Methods The linear ubiquitin substrate protein His-Ub-Ub-Flag was designed, expressed, and purified. Using OTULIN80-352 as the target enzyme and PR619 as a positive control, the enzyme-to-substrate ratio and reaction time were optimized to establish an in vitro OTULIN activity evaluation system. Results The linear ubiquitin substrate protein His-Ub-Ub-Flag was successfully obtained. OTULIN activity was significantly inhibited by PR619, which confirmed the feasibility of the assay. The optimal enzyme-to-substrate ratio of OTULIN80-352 to His-Ub-Ub-Flag was 1∶16, and the optimal reaction condition was incubation at 37℃ for 20 min. Conclusion An in vitro enzymatic activity evaluation system for OTULIN was successfully established, which could be used to evaluate the inhibitory effects of candidate compounds on OTULIN activity and provide technical support for subsequent screening of OTULIN small-molecule inhibitors.
