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重组FLAG标签的筛选、鉴定及与新型荧光蛋白的融合表达

发表时间:2010-10-15  浏览量:2249  下载量:1062
全部作者: 易科,高晓莲
作者单位: 浙江理工大学生命科学学院;中南大学湘雅医院中国医学遗传学国家重点实验室
摘 要: 以FLAG标签的原始序列DYKDDDDK(FLAG4)为模板,设计4种新FLAG标签与新型绿色荧光蛋白(G2)、红色荧光蛋白(H10)融合。Western blotting分析表明,抗FLAG单克隆抗体M2(AFM2)能够特异识别G2-FLAG4,G2-FLAG5,H10-FLAG3,H10-FLAG4,H10-FLAG5等5种FP-FLAG融合蛋白。采用非竞争性ELISA固相法,计算出FP-FLAG融合蛋白与单克隆抗FLAG M2抗体(AFM2)的亲和常数。结果表明,FLAG原始序列与AFM2之间的亲和力最高,FLAG5和FLAG3次之,FLAG1和 FLAG2不能与AFM2特异识别。在FLAG融合蛋白的纯化应用中,运用这些FLAG标签的亲和力差异,在同一纯化体系下通过设计不同的洗提次序和洗提条件,可以达到逐步分离不同目的物的效果。
关 键 词: 多肽与蛋白质生物化学;重组FLAG标签;新型荧光蛋白;非竞争性ELISA;亲和常数
Title: Construction, expression and characterization of fusion proteins consisting of novel fluorescent protein and recombinant FLAG tag
Author: YI Ke, GAO Xiaolian
Organization: College of Life Science, Zhejiang Sci-Tech University; State Key Laboratory of Medical Genetics of China, Xiangya Medical School, Central South University
Abstract: Based on FLAG-tag initial sequence (FLAG4: DYKDDDDK), designed four new FLAG tags and fused with H10 and G2. Western blotting was employed and the result showed that monoclonal antibody ANTI-FLAG M2 (AFM2) interacted specifically with five kinds of FP-FLAG fusion protein(G2-FLAG4, G2-FLAG5, H10-FLAG3, H10-FLAG4, H10-FLAG5).The functional affinity of FP-FLAG fusion protein and monoclonal antibody ANTI-FLAG M2 (AFM2) was determined by non-competitive ELISA. Results show that AFM2 against FLAG-tag initial sequence have the highest affinity, DYKDEDDK, DYKDYKDYK take the second place and DYKGGGYK is incapable of binding the antibody of AFM2. Using different affinity of these FLAG-tags, by designing different elution sequence and elution condition various FLAG fusion proteins can be gradually purified.
Key words: peptide and protein biochemistry; recombination FLAG-tag; novel fluorescent proteins; non-competitive ELISA; affinity constant
发表期数: 2010年10月第19期
引用格式: 易科,高晓莲. 重组FLAG标签的筛选、鉴定及与新型荧光蛋白的融合表达[J]. 中国科技论文在线精品论文,2010,3(19):1983-1991.
 
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