真核表达肝药酶UGT1A9可溶重组蛋白方法探索  

Exploration of Solution Recombinant Protein Method of Hepatic Drug-Metabolizing Enzyme UGT1A9 in Eukaryotic Cells

作　　者：尹鑫俐 戚旭丹 涂予溪 陆茜 杨元 陈侠斌 童骏森 YIN Xinli;QI Xudan;TU Yuxi;LU Xi;YANG Yuan;CHEN Xiabin;TONG Junsen(School of Pharmacy,Hangzhou Normal University,Hangzhou 311121,China)

机构地区：[1]杭州师范大学药学院,浙江杭州311121

出　　处：《杭州师范大学学报（自然科学版）》2023年第4期351-357,共7页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：国家自然科学基金项目(32101010).

摘　　要：UDP-葡萄糖醛酸基转移酶(UGTs)是人体重要的肝脏药物代谢酶,其底物选择性、催化机制等研究因难以获得可溶性全酶而受到限制.本研究以制备UGT1A9可溶重组蛋白为研究目标,通过筛选重组蛋白表达体系、融合标签种类及linker长度,优化重组蛋白表达纯化条件和方法,实现了重组UGT1A9的大量制备.HEK293F细胞分泌表达的His8-MBP-NSAS-UGT1A9(氨基酸25-466)重组蛋白经Ni-TED亲和纯化介质从培养基上清液中分离,洗脱产物经SDS-PAGE、Western blot及MS检测鉴定为目的蛋白,通过Bradford法测得重组蛋白最终产量为2 mg/L,纯度达到95%以上.本研究建立了一种人源药物代谢酶UGT1A9的可溶重组蛋白表达纯化方法,为UGT1A9及UGTs同工酶的药物代谢、催化机制及结构解析研究奠定基础.UDP-glucuronosyltransferase(UGTs)enzymes are major human hepatic drug-metabolizing enzymes.However,their substrate selectivity and catalytic mechanism have been restrained due to the challenge of soluble recombinant protein preparation.This study aimed to obtain soluble UGT1A9 recombinant protein,and achieved the large-scale preparation of recombinant UGT1A9 by investigating the effects of diverse recombinant protein expression systems,fusion proteins,linker length,and purification conditions and methods.The soluble recombinant His 8-MBP-NSAS-UGT1A9(amino acids:25-466)protein was isolated from the supernatant of HEK293F cells culture through affinity chromatography by Ni-TED resins.After washing,the eluent containing target protein was collected and concentrated for SDS-PAGE,Western blot,and mass spectrometry(MS)analysis.The final yield of target protein determined by Bradford method was approximately 2 mg/L,with a purity more than 95%.The study established a soluble expression and purification process for human UGT1A9,which could lay a foundation for the subsequent studies of drug metabolism,catalytic mechanism,and structure analysis of UGT1A9 and other UGT isoenzymes.

关 键 词：药物代谢酶 UDP-GLUCURONOSYLTRANSFERASE UGT1A9 真核表达 蛋白纯化 

分 类 号：R915[医药卫生—微生物与生化药学]