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    • 4. 发明申请
    • COMBINATORIAL PROTEIN LIBRARY SCREENING BY PERIPLASMIC EXPRESSION
    • 通过周边表达组合蛋白图书馆筛选
    • WO2005019409A2
    • 2005-03-03
    • PCT/US2003/021928
    • 2003-07-15
    • BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEMHARVEY, Barrett, R.GEORGIOU, GeorgeIVERSON, Brent, I.
    • HARVEY, Barrett, R.GEORGIOU, GeorgeIVERSON, Brent, I.
    • C12N
    • C07K16/005C07K2317/622C07K2317/92C12N15/1034C12Q1/025G01N33/6845
    • 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. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in the periplasm of gram negative bacteria and 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 outer surface-expression of ligand fusion proteins employed with phage display. The inventors have also provided improved antobodies that were initially prepared using the sceening methods developed.
    • 本发明通过提供用于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)的文库在革兰氏阴性细菌的周质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,并且避免了与噬菌体展示一起使用的配体融合蛋白的外表面表达相关的问题。 本发明人还提供了改进的本体,其最初使用所开发的方法制备。