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    • 7. 发明授权
    • Circularly permuted biotin binding proteins
    • 循环置换的生物素结合蛋白
    • US06492492B1
    • 2002-12-10
    • US09285867
    • 1999-04-02
    • Patrick S. Stayton
    • Patrick S. Stayton
    • C07K1400
    • C07K14/36C07K2299/00C07K2319/00C07K2319/22C12N15/62Y10S530/825
    • Circularly permuted proteins are described wherein the natural termini of the polypeptide are joined and the resulting circular protein is opened at another point to create new C- and N- termini. The resulting protein exhibits some altered characteristic such as reduced substrate binding, for example. Fusion proteins can be made from the circularly permuted protein by attaching the second polypeptide to these newly created termini. These fusion proteins will have altered properties from a fusion protein made by attaching the second polypeptide to the natural termini. For example, the second peptide or protein can be attached at a position where it is more accessible to its substrate or intended target. In the preferred embodiment, the base circularly permuted biotin binding protein. In one embodiment, a flexible polypeptide loop important for the binding of biotin was opened by creation of the circularly permuted protein. The original termini (residues 13 and 139 of SEQ ID NO:1) were joined by a linker. The biotin association constant was reduced approximately six orders of magnitude below that of wild type streptavidin to 107 M−1. Fusion proteins of the circularly permuted streptavidin can be made with secondary peptides/proteins such as IgG binding protein A or single-chain antibodies.
    • 描述了循环置换的蛋白质,其中多肽的天然末端连接并且得到的环状蛋白质在另一个位置打开以产生新的C-和N-末端。 所得到的蛋白质例如显示出一些改变的特征,例如降低的底物结合。 融合蛋白可以通过将第二多肽连接到这些新产生的末端从循环置换的蛋白质制备。 这些融合蛋白将具有通过将第二多肽连接到天然末端而制备的融合蛋白质的改变的性质。 例如,第二个肽或蛋白质可以连接到其底物或预期靶标更容易接近的位置。 在优选的实施方案中,碱基环状置换的生物素结合蛋白。 在一个实施方案中,通过产生循环置换的蛋白质来打开对生物素结合重要的柔性多肽环。 原始末端(SEQ ID NO:1的残基13和139)通过接头连接。 生物素缔合常数比野生型链霉抗生物素蛋白降低约六个数量级至107 M-1。 可以用二级肽/蛋白质例如IgG结合蛋白A或单链抗体制备环状置换的链霉抗生物素蛋白的融合蛋白。