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    • 2. 发明申请
    • ISOLATION OF BINDING PROTEINS WITH HIGH AFFINITY TO LIGANDS
    • 分离与配体具有高度亲和力的结合蛋白
    • WO0234886A9
    • 2003-02-13
    • PCT/US0146795
    • 2001-10-26
    • UNIV TEXASCHEN GANGHAYHURST ANDREWTHOMAS JEFFREY GIVERSON BRENT LGEORGIOU GEORGE
    • CHEN GANGHAYHURST ANDREWTHOMAS JEFFREY GIVERSON BRENT LGEORGIOU GEORGE
    • C12N15/09C12N15/10C12N5/00
    • C12N15/1034
    • The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides via "display-less" library screening. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in soluble form in the periplasmic space of gram negative bacteria, such as Escherichia coli, and are mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the surface-expression of ligand fusion proteins employed with phage display.
    • 本发明通过提供一种通过“无需显示”文库筛选来分离能够结合小分子和肽的结合蛋白的快速方法克服了现有技术的不足。 在该技术中,候选结合蛋白如抗体序列的文库以可溶形式在革兰氏阴性细菌如大肠杆菌的周质空间中表达,并与标记的配体混合。 在表达对配体具有亲和性的重组多肽的克隆中,与结合蛋白结合的标记配体的浓度增加并且允许从文库的其余部分分离细胞。 在使用靶标配体的荧光标记的情况下,可以通过荧光激活细胞分选(FACS)分离细胞。 该方法比现有技术方法更快并且避免了与噬菌体展示中使用的配体融合蛋白的表面表达相关的问题。
    • 4. 发明申请
    • ISOLATION OF BINDING PROTEINS WITH HIGH AFFINITY TO LIGANDS
    • 分离具有高亲和力的结合蛋白
    • WO0234886A3
    • 2003-07-31
    • PCT/US0146795
    • 2001-10-26
    • UNIV TEXASCHEN GANGHAYHURST ANDREWTHOMAS JEFFREY GIVERSON BRENT LGEORGIOU GEORGE
    • CHEN GANGHAYHURST ANDREWTHOMAS JEFFREY GIVERSON BRENT LGEORGIOU GEORGE
    • C12N15/09C12N15/10C12N5/00
    • C12N15/1034
    • The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides via "display-less" library screening. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in soluble form in the periplasmic space of gram negative bacteria, such as Escherichia coli, and are mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the surface-expression of ligand fusion proteins employed with phage display.
    • 本发明通过提供通过“无显示”文库筛选分离能够结合小分子和肽的结合蛋白的快速方法来克服现有技术的缺陷。 在该技术中,候选结合蛋白(例如抗体序列)的文库以可溶形式表达在革兰氏阴性细菌如大肠杆菌的周质空间中,并与标记的配体混合。 在表达对配体具有亲和性的重组多肽的克隆中,与结合蛋白结合的标记配体的浓度增加,并允许细胞与文库的其余部分分离。 当使用靶配体的荧光标记时,可以通过荧光激活细胞分选(FACS)分离细胞。 该方法比现有技术方法更快速,并避免与噬菌体展示所用配体融合蛋白的表面表达相关的问题。