绿色荧光蛋白结合蛋白的表达纯化与鉴定  

Expression,Purification and Characterization of Green Fluorescent Protein Binding Protein

作　　者：胡业辉 李益鹏 王紫英 陈勖 任真 丁澦[1] HU Yehui;LI Yipeng;WANG Ziying;CHEN Xu;REN Zhen;DING Yu(Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200438, China)

机构地区：[1]复旦大学生命科学学院生理学与生物物理学系,上海200438

出　　处：《复旦学报（自然科学版）》2019年第2期183-190,共8页Journal of Fudan University：Natural Science

基　　金：国家自然科学基金(31470764);国家基础学科拔尖学生培养试验计划

摘　　要：绿色荧光蛋白(GFP)及其衍生物已经被广泛应用于生物化学和分子生物学研究,其纳米抗体(GBP)也已在近期被鉴定,但GBP大规模表达及纯化流程优化尚未见报道,并且GBP与不同荧光蛋白之间的亲和力尚未被测定.我们首先构建了GBP基因(GBP1)的原核表达系统,然后通过Ni-NTA亲和层析和Mono Q阴离子交换层析纯化,每升菌液能够得到超过43mg、纯度超过95%的重组GBP1蛋白.我们通过MALDI-TOF/TOF质谱法测定纯化的GBP1的精确分子量,通过微量热泳动(MST)和等温滴定量热(ITC)实验检测GBP1与7种不同来源荧光蛋白的亲和力.结果显示GBP1特异性地与源自Aequorea victoria的荧光蛋白及其衍生荧光蛋白相互作用,与RFP和Zoanthus GFP等无相互作用.GBP1与EGFP和sfGFP的亲和力最高,与YPet和T-Sapphire的亲和力稍弱,与CyPet的亲和力最低.Green fluorescent protein (GFP) and its derivatives are widely used in biochemistry and molecular biology research. Recently, GFP binding protein (GBP) was developed from camel antibody s heavy chain variable domain. However, the binding ability of GBP to other fluorescent proteins was not verified, and the large scale production of GBP was not optimized. In this work, we first synthesized the GBP gene (GBP1) and cloned GBP1 into a prokaryotic expression system. The recombinant GBP1 was purified by sequential Ni-NTA affinity chromatography and Mono Q anion exchange chromatography. The purity of GBP1 was over 95 % and the yield was over 43 mg per liter culture. The molecular weight of the purified GBP1 was verified by linear mode MALDI-TOF/TOF mass spectrometry. The binding of GBP1 to a series of fluorescent proteins was tested by size exclusive chromatography, microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) assay. The results showed that GBP1 specifically interacted with fluorescent proteins derived from Aequorea victoria but didn t bind to RFP or Zoanthus GFP. The binding affinity of GBP1 to EGFP or sfGFP was high, its affinity to YPet or T-Sapphire was moderate, and its affinity to CyPet was relatively weak.

关 键 词：荧光蛋白 绿色荧光蛋白结合蛋白 纳米抗体 蛋白表达 蛋白纯化 微量热泳动 

分 类 号：Q513[生物学—生物化学]