基于FRET原理的TEV酶活性检测方法的探索  

FRET-based Activity Detection System of TEV Protease

作　　者：王婧瑶 祝佳慧 续敏 王盛 李家璜 华子春 WANG Jing-yao;ZHU Jia-hui;XU Min;WANG Sheng;LI Jia-huang;HUA Zi-chun(School of Biopharmacy,China Pharmaceutical University,Nanjing 211198,China;School of Life Sciences,Nanjing University,Nanjing 210023,China;Institute of Pharmaceutical Biotechnology of Jiangsu Industrial Technology Research Institute,Changzhou 213164,China;Nanjing Genrecom Laboratories,Ltd.,Nanjing 210032,China)

机构地区：[1]中国药科大学生物药物学院,江苏南京211198 [2]南京大学生命科学学院,江苏南京210023 [3]江苏省产业技术研究院医药生物技术研究所/常州南京大学高新技术研究院,江苏常州213164 [4]南京吉芮康生物科技研究院,江苏南京210032

出　　处：《药物生物技术》2024年第1期1-8,共8页Pharmaceutical Biotechnology

基　　金：国家自然科学基金(No.32250016,No.32171460);江苏省科技厅项目(No.BK20230165,No.BE2023695,No.BK20231136);常州市科技局项目(No.CJ20230017,No.CJ20220019,No.CJ20235009)。

摘　　要：文章开发了一种基于荧光蛋白的FRET检测技术,探索其在研究TEV蛋白酶活性等方面的应用。在青色荧光蛋白(Cerulean)和黄色荧光蛋白(Citrine)之间插入TEV酶特异性识别位点,构建基于FRET原理的重组底物。在细菌内、外两个体系中,通过酶切前后SDS-PAGE分析及底物荧光强度的变化,对TEV蛋白酶活性的差异进行表征,以此验证该方法的可行性。随着酶含量的增加,基于FRET原理的底物被切割效果明显增强。且在细菌内、细菌外两个体系中,阳性对照均显示出更低的荧光强度。蛋白酶类FRET底物可以在细菌内和细菌外完成对野生型TEV酶及变体的酶活比较,揭示了其在酶活检测以及筛选方面的应用前景,并为进一步开发其他蛋白酶活性检测技术提供了参考和借鉴。Fluorescence resonance energy transfer(FRET)is a non-radiative energy transfer phenomenon based on the dipole-dipole coupling between a fluorophore donor and a fluorophore acceptor.FRET technology based on fluorescent protein has been widely used in biotechnology.In this paper,a FRET detection technology based on fluorescent protein was developed,and its application in the study of protease activity was discussed.The fluorescent protein recombinant substrate based on the FRET principle for detecting protease activity was constructed.The difference of TEV protease activity was characterized by the change of FRET fluorescence intensity.The pET 28a-cerulean-substrate-linker-citrine plasmid was constructed by inserting the TEV protease specific recognition sites between the sequences of citrine and cerulean.The changes of fluorescence intensity by inducing the expression of FRET substrate and TEV protease in bacteria were observed.After the FRET substrate and TEV protease were purified,the reaction system was established to observe the change of fluorescence intensity after enzymatic digestion.The cleavage effect of FRET substrate was significantly enhanced with the increase of TEV protease content.The positive control showed lower fluorescence intensity in both the intra bacterial and extra bacterial systems.It was proved that the cutting efficiency of the positive control was higher than that of the wild type.It showed that the FRET substrate was successfully constructed,and the comparison of TEV protease activity could be completed through the changes in fluorescence intensity both inside and outside the cell.FRET substrate could complete the comparison of the enzyme activities of wild-type TEV protease and variants in both the internal and external bacterial systems.And it could be used in the activity screening of TEV protease in the future.The method revealed its application prospect in enzyme activity detection,laying a foundation for the subsequent exploration of enzymatic properties,and providing a reference

关 键 词：TEV酶 荧光共振能量转移技术(FRET) 重组蛋白 蛋白表达与纯化 青色荧光蛋白 黄色荧光蛋白 

分 类 号：Q51[生物学—生物化学]