基于抗坏血酸过氧化酶的电镜成像和活细胞邻近标记方法的应用进展  

Advances in Applications of Ascorbate Peroxidase in Electron Microscope Imaging and Proximity Labeling in Live Cells

作　　者：徐晓君 朱新宇 张蕊 薛艳红[2] 李真珍[3] 宋婀莉[2] 侯俊杰 XU Xiao-Jun,ZHU Xin-Yu,ZHANG Rui,XUE Yan-Hong,LI Zhen-Zhen,SONG E-Li,HOU Jun-Jie(1. College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China, 2.National Laboratory of Biomacromolecules,lnstitute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, 3. Nephrology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Chin)

机构地区：[1]华中科技大学生命科学与技术学院,武汉430074 [2]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101 [3]郑州大学第一附属医院肾内科,郑州450052

出　　处：《生物化学与生物物理进展》2018年第5期519-528,共10页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(31770891;31400703;31770900)~~

摘　　要：Alice Ting实验室开发的抗坏血酸过氧化物酶(engineered ascorbate peroxidase,APEX),相对于经典的辣根过氧化物酶(horse radish peroxidase,HRP),其酶活性不再受细胞内蛋白质定位的影响,可以在几乎所有的亚细胞区域保持活性,这使其在研究亚细胞尺度以及活细胞水平生物学问题时极具优势.目前,基于APEX的二氨基联苯胺(diaminobenzidine,DAB)染色标记技术已经成功地实现对全细胞、亚细胞器和蛋白质水平的电镜成像.同时,与质谱技术结合,基于APEX的活细胞生物素邻近标记方法也极大地推动了亚细胞器蛋白质组学,以及目标蛋白在特定时空条件下邻近蛋白质组学的研究发展.本文将从以上两个方面阐述APEX技术的基本原理及最新应用进展,并讨论和展望其在实际应用中存在的局限性和挑战.The engineered ascorbate peroxidase（APEX） developed by Alice Ting laboratory, compared with classical horse radish peroxidase（HRP）, can keep active within all cellular compartments, so it has great potential as a tool for studying the fusion proteins in both subcellular organelles and live cells. Up to now, diaminobenzidine staining based on APEX tag has been successfully developed for electron microscopy（EM） in whole cells,subcellular organelles and proteins. Moreover, combined with mass spectrometry technique, APEX-mediated proximity biotin labeling in living cells greatly promoted the study of subcellular organelle proteomics and temporal-spatial proteomics. This review focused on the principle of APEX methodology, summarized its latest applications including EM imaging and spatial proteomics, and discussed its limitations and challenges as well.

关 键 词：抗坏血酸过氧化物酶 二氨基联苯胺染色标记 电镜 邻近标记 蛋白质组 蛋白质相互作用 

分 类 号：R6[医药卫生—外科学] R7[医药卫生—临床医学]