Binary polypeptide system for permanent and oriented protein immobilization
Binary polypeptide system for permanent and oriented protein immobilization
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DOI：
10.1186/1477-3155-8-9
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发表时间：
2010-05-12
影响因子：
10.2
通讯作者：
Davletov, Bazbek
中科院分区：
文献类型：
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作者：
Ferrari, Enrico;Darios, Frederic;Davletov, Bazbek
背景：分子生物学、临床诊断和生物技术中的许多技术依赖于二元亲和标签。现有的标签是基于小分子（例如，例如，在一个实施例中，生物素/链霉亲和素或谷胱甘肽/GST）或肽标签（FLAG、Myc、HA、Strep-标签和His-标签）。其中，生物素-链霉亲和素系统是最受欢迎的，因为生物素与四聚体蛋白链霉亲和素几乎不可逆的相互作用。然而，稳定的生物素-链霉抗生物素蛋白系统的主要缺点是这两个标签都不能通过重组方法添加到感兴趣的蛋白质中（除了Strep标签的情况）导致需要化学偶联。在这里，我们报告了一种新的固定化系统，该系统利用两个单体多肽自组装产生非共价但几乎不可逆的复合物，该复合物在强洗涤剂中稳定，离液剂，以及酸和碱。我们的系统是基于被称为SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）复合物的四螺旋束的核心区域。这种不可逆蛋白质附着系统（IPAS）使用缩短的突触融合蛋白螺旋和融合的SNAP 25-突触泡蛋白或融合的突触融合蛋白-突触泡蛋白和SNAP 25，从而形成适合重组蛋白标记、捕获和固定的双组分系统。我们还表明，IPAS是适合使用传统的珠和色谱，平面表面和Biacore，金纳米粒子和蛋白质-蛋白质相互作用在solution.Conclusions：IPAS提供了一种替代化学交联，链霉亲和素-生物素系统和传统的肽亲和标签，并可用于广泛的应用在纳米技术和分子科学。
Background: Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e. g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling.Results: Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution.Conclusions: IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.