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azurin荧光探针的构建、表达及性质研究

发表时间:2013-03-15  浏览量:1085  下载量:506
全部作者: 张茜,杨弋
作者单位: 华东理工大学药学院,生物反应器工程国家重点实验室
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,以及其附近配位氨基对蛋白分子构象的影响,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一系列插入位点,将其与一种发射波长在600 nm附近的红色荧光蛋白mcherry融合。然后,在大肠杆菌中诱导表达并纯化融合蛋白。通过检测发现:融合蛋白具有红色荧光,不同氧化还原条件下,融合蛋白荧光发生相应变化,表明已成功构建基于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: 生物化学;天青素蛋白;红色荧光蛋白;氧化还原;铜;荧光探针
Title: Research on construction, expression and characteristics of the fluorescent sensor based on azurin
Author: ZHANG Qian, YANG Yi
Organization: State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology
Abstract: As one of the copper blue protein family member, azurin is sensitive to oxidation and reduction. In order to further understanding the role of copper atomic as active center in azurin and the influence of the coordination of amino on the protein conformation, AZURIN gene was cloned for the construction of azurin protein restructuring plasmid, then we selected series sites nearby copper coordination center. The plasmid was fused into a red fluorescent protein mcherry whose excitation and emission wavelength is nearby 600 nm. The fusion protein was expressed in E.coli BL21 (DE3) and then was purified. It was found that the fusion protein had red fluorescence, and the fluorescence changed in different redox conditions, indicating redox sensitive fluorescence probe based on azurin was constructed successfully. This research provides an effective tool for the further exploration on azurin protein conformation of activity center and its sensitive environment.
Key words: biochemistry; azurin; mcherry; redox; copper; fluorescent sensor
发表期数: 2013年3月第5期
引用格式: 张茜,杨弋. azurin荧光探针的构建、表达及性质研究[J]. 中国科技论文在线精品论文,2013,6(5):407-413.
 
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