基于Nanoluc技术和荧光分析技术建立蛋白质水解靶向嵌合体筛选方法及评价  

PROTAC Screening Method and Evaluation Based on Nanoluc and Fluorescence Analysis Techniques

作　　者：刘明秋 吴波 吴正升[1] 张令强[1,2] 崔春萍 LIU Ming-Qiu;WU Bo;WU Zheng-Sheng;ZHANG Ling-Qiang;CUI Chun-Ping(School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;Institute of Biomics,Academy of Military Medicine,Academy of Military Sciences,National Center for Protein Science(Beijing),Beijing 102200,China)

机构地区：[1]安徽医科大学基础医学院,合肥230032 [2]军事科学院军事医学研究院生命组学研究所,国家蛋白质科学中心(北京),北京102200

出　　处：《生物化学与生物物理进展》2023年第4期841-849,共9页Progress In Biochemistry and Biophysics

基　　金：国家重点研发计划(2021YFA1300200);国家自然科学基金(82192884)资助项目。

摘　　要：目的为了从一系列旨在降解靶蛋白的化合物中筛选出高效的蛋白质水解靶向嵌合体(PROTAC),本文建立了一个稳定的高通量PROTAC筛选方法。方法Nanoluc荧光素酶有LgBiT和HiBiT两个亚基组成,通过将HiBiT标签与mCherry(红色荧光蛋白)、目的蛋白、Halo标签融合表达,LgBiT与GFP(绿色荧光蛋白)融合表达,利用GFP与mCherry的共定位情况可直观评价Nanoluc荧光素酶的组装情况,而通过监测Nanoluc的活性可以指示目的蛋白的含量。利用慢病毒包装系统构建稳定过表达GFP-LgBiT和HiBiT-mCherry-Target-Halo的细胞系,使用可募集Halo标签融合蛋白被Cul2-Rbx1-Elo BCVHL复合体降解的Halo PROTAC3诱导HiBiT-mCherry-Target-Halo降解,进一步利用蛋白质免疫印迹(Westernblot)、Nanoluc荧光素酶活性分析系统和流式细胞术分别评价Halo PROTAC3诱导底物降解的效率。结果Halo PROTAC3高效降解HiBiT-mCherry-Target-Halo,并呈现浓度和时间依赖性。结论本文建立了一种联合Nanoluc技术和荧光分析的PROTAC筛选策略,用Halo PROTAC3作为阳性对照,可以快速评价PROTAC分子使底物蛋白被降解的效率,实现对PROTAC的“优化选择”或者“优中选优”,为最终PROTAC的开发应用提供保障。Objective To screen efficient PROTAC from a bound of compounds designed to degrade a specific target,we established a stable and high-throughput screening system.Methods The two subunits of Nanoluc,HiBiT and LgBiT,was fused with mCherry-target-Halo and GFP,respectively.Colocalization of mCherry and GFP indicates the well-assembly of Nanoluc,and the activity of Nanoluc reflects the amount of target protein directly.Stable cell lines overexpressing HiBiT-mCherry-Target-Halo and GFP-LgBiT were constructed using lentivirus packaging systems.HaloPROTAC3,which recuits Halo tagged protein to Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex,was used to induce the degradation of Halo tag fused target protein.Western blot,Nanoluc activity analysis,and flow cytometry were used to evaluate the degradation efficiency of HaloPROTAC3.Results HaloPROTAC3 induce the degradation of HiBiT-mCherry-Target-Halo effectively in a time and concentrition dependent manner.Conclusion A novel strategy combined with Nanoluc and fluorescence analysis for PROTAC screening was developed.With HaloPROTAC3 as a positive control,the strategy provides a guarantee for the optimal development and application of PROTAC.

关 键 词：蛋白质水解靶向嵌合体 降解剂 筛选 Nanoluc 

分 类 号：Q2-33[生物学—细胞生物学]