Azurin荧光探针的构建、表达及性质研究.doc

  Azurin 荧光探针的构建、表达及性质研究# 张茜，杨弋** （华东理工大学药学院，生物反应器工程国家重点实验室，上海 200237） 5 10 摘要：天青素蛋白 Azurin 是一种对氧化还原敏感的铜蓝蛋白。为进一步了解铜原子作为 Azurin 活性中心的关键成分及其附近配位氨基对蛋白分子构象的影响，我们将 Azurin 基因 克隆至原核载体构建 Azurin 蛋白重组质粒，然后在铜配位中心附近选择了一系列插入位点, 将其与一种激发发射波长在 600nm 附近的红色荧光蛋白 mcherry 融合。我们在大肠杆菌中诱 导表达并纯化了这些融合蛋白。这些蛋白质具有红色荧光,不同氧化还原条件下，融合蛋白 荧光发生了相应的变化。这些研究结果表明我们成功构建了基于 Azurin 的氧化还原敏感的 荧光探针，这为进一步探究 Azurin 蛋白活性中心分子构象及其环境敏感性提供了有效的工 具。 关键词：天青素蛋白；红色荧光蛋白；氧化还原；铜；荧光探针 中图分类号：Q51 15 Recombinant construction、expression and characteristics of the fluorescent sensor based on Azurin Zhang Qian, Yang Yi (State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of 20 25 30 35 40 Science and Technology, ShangHai 200237) Abstract: As one of the copper blue protein family member, Azurin is sensitive to oxidation and reduction. In order to further understanding the influence to the Azurin conformation by the copper atomic and the coordination of protein molecules amino nearly, for the first the Azurin gene was cloned for the construction of Azurin protein restructuring plasmid, then combined with copper coordination center conformation,we selected series sites and choosed a red fluorescent protein mcherry whose excitation and emission wavelength is nearby the 600 nm to fusion. We expressed those fusion protein in E.coli BL21 (DE3), these protein has red fluorescent, and fusion proteins fluorescence has a corresponding change in different redox conditions. The results show that we have successfully constructed redox sensitive fluorescence probe based on Azurin.This development provides a effective tool for the further exploration of Azurin protein molecular conformation near activity center and its environmental sensitivity. Keywords: Azurin; mcherry; redox; copper; fluorescent sensor 0 引言 早在二十世纪九十年代，研究者对于该广泛存在于细菌和植物体内起电子传递作用的铜 蓝蛋白就有着浓厚兴趣，而 Adman,E.T 等人首先通过 X-射线晶体学方法得到了不同细菌来 源的 Azurin 三维构象[1-2]。天然 Azurin 三维构象呈三明治状，含有一个铜原子，由 8 个 β 折 叠链形成的两片拓扑桶状结构将铜原子夹在中间，铜配位中心既为 Azurin 活性中心同时也 是氧化还原位