Määttä Juha A E, Helppolainen Satu H, Hytönen Vesa P, Johnson Mark S, Kulomaa Markku S, Airenne Tomi T, Nordlund Henri R
Institute of Medical Technology, Biokatu 6, FI-33014 University of Tampere and Tampere University Hospital, Tampere, Finland.
BMC Struct Biol. 2009 Sep 29;9:63. doi: 10.1186/1472-6807-9-63.
Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog.
Xenavidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin.
The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.
抗生物素蛋白是一类具有极高配体结合亲和力的蛋白质，这种特性在众多生命科学应用中都有广泛应用。尽管抗生物素蛋白对生物技术和纳米技术很有用，但其生物学功能尚未完全明确。在此，我们对一种名为非洲爪蟾抗生物素蛋白（xenavidin）的新型抗生物素蛋白进行了结构和功能表征，据我们所知，这是首次报道的来自青蛙的抗生物素蛋白。
从热带爪蟾的EST序列数据库中鉴定出非洲爪蟾抗生物素蛋白，并利用杆状病毒表达系统在昆虫细胞中生产。基于凝胶过滤分析，发现重组非洲爪蟾抗生物素蛋白为同四聚体。使用Biacore传感器分析、荧光标记生物素和放射性生物素评估非洲爪蟾抗生物素蛋白的生物素结合特性——它与生物素具有高亲和力，但结合程度不如鸡抗生物素蛋白和细菌链霉抗生物素蛋白紧密。X射线晶体学揭示了配体结合位点周围的结构保守性，而一些环区具有独特设计。配体结合位点入口处和/或内部结构水分子的位置可能在决定非洲爪蟾抗生物素蛋白独特的生物素结合特性中发挥作用。
本文报道的新数据提供了有关生物素结合的生化和结构重要决定因素的信息。这些信息可能有助于发现新型生物技术工具。
