溶酶体代谢酶甘露糖-6-磷酸糖基化修饰关键酶UCE的异源表达及功能鉴定  

Expression and characterization of UCE,a key enzyme of lysosomal hydrolase to synthesize mannose-6-phosphate

作　　者：曹筠嵩 杨燕 刘忞之 王伟 CAO Yun-song;YANG Yan;LIU Min-zhi;WANG Wei(State Key Laboratory of Bioactive and Function of Natural Medicines&Key Laboratory of Biosynthesis of Natural Products,Institute of Materia Medica,Chinese Academy of Medical Science&Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室/国家卫生和计划生育委员会天然药物生物合成重点实验室,北京100050

出　　处：《中国医药生物技术》2022年第6期481-487,共7页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(82073757、81903487);中国医学科学院医学与健康科技创新工程(2021-I2M-1-029);北京协和医学院合成生物学学科建设专项(201920100801)。

摘　　要：目的 利用大肠杆菌表达系统对溶酶体代谢酶甘露糖-6-磷酸(M6P)糖基化修饰的关键酶N-乙酰葡萄糖胺-1-磷酸二酯α-N-乙酰葡萄糖胺糖苷酶(UCE)进行异源表达和功能鉴定。方法 通过UCE的氨基酸序列分析,分别截去UCE的信号肽、前体肽、跨膜区、胞浆尾肽等结构域,形成不同形式的截短型UCE。利用促溶标签麦芽糖结合蛋白(MBP)的载体和大肠杆菌表达系统,对不同的截短型UCE进行表达。将表达的可溶性融合蛋白利用Ni离子琼脂糖凝胶进行亲和层析纯化,并利用UDP-Glo试剂盒测定融合蛋白的活性。结果 截短型的UCE-MBP融合蛋白能够被可溶性地融合表达,并且能够利用Ni离子琼脂糖凝胶进行亲和层析纯化。去掉其前体肽和仅保留核心区的UCE融合蛋白能够展现出较高的活性。结论 利用促溶标签蛋白能够实现UCE在大肠杆菌中可溶性地融合表达,从而能够快速制备大量的UCE,该酶可用于对溶酶体代谢酶体外糖基化的生物催化修饰。Objective To express and purify N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase(UCE), one of the key enzymes of mannose-6-phosphate glycosylation modification pathway, and then determine the activity of UCE.Methods According to the canonical UCE structure, the signal peptide, propiece, transmembrane domain and cytoplasmic tail of UCE was truncated, respectively, to construct the different mutants of UCE. A maltose binding protein(MBP) tag was fused to the N-terminals of differently truncated UCEs. The resulting expression plasmids were transformed into Escherichia coli Shuffle T7, and then induced to express. The expressed fusion protein was purified by Ni-agrose gel affinity chromatography from the soluble supernatant of total proteins, and the enzyme activity was determined according to the manual of UDP-Glo kit.Results The truncated MBP-UCE fusion proteins were able to be solubly expressed, and purified by Ni-agrose gel affinity chromatography. The results of enzymatic assay indicated that the UCE fusion proteins with propiece truncated and only the core remained showed higher activity.Conclusion The active UCE proteins with the fused MBP-tag were solubly expressed in E. coli, and a large amount of them could be prepared in a short time by affinity chromatography. It will be beneficial to contribute an in vitro enzymatic glycosylation modification of lysosomal hydrolases.

关 键 词：溶酶体代谢酶 N-糖基化 N-乙酰葡萄糖胺-1-磷酸二酯α-N-乙酰葡萄糖胺糖苷酶 异源表达 

分 类 号：Q78[生物学—分子生物学]