Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University Nanjing 210093.
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University Nanjing 210093; Institute of Molecular & Experimental Therapeutics, East China Normal University, Shanghai 200062.
J Biol Chem. 2010 Mar 26;285(13):9367-9372. doi: 10.1074/jbc.M109.059857. Epub 2010 Jan 29.
To find a suitable ligand from a complex antigen system is still a mission to be accomplished. Here we have explored a novel "library against proteome" panning strategy for ligand screening and antigen purification from a complex system using phage-displayed antibody technology. Human plasma proteome was targeted for phage library panning. During the process, the panning was carried out in solution, using a biotin/streptavidin beads separation system, for three rounds. Nine monoclonal phages, bound tightly to a number of unknown plasma proteins, were selected from the last round, six of which were directly employed as cross-linked matrices to purify their corresponding antigens from the plasma. The proteins isolated by G5 and E1 matrices were identified as amyloid protein and apolipoprotein A-I precursor, respectively. The results demonstrated that it was feasible to simultaneously obtain a number of ligand phages for various antigens, including low abundant proteins in a non-comparative proteome-wide system.
从复杂的抗原系统中找到合适的配体仍然是一项有待完成的任务。在这里，我们探索了一种新的“针对蛋白质组的文库”淘选策略，用于使用噬菌体展示抗体技术从复杂系统中筛选配体和纯化抗原。以人类血浆蛋白质组为靶标进行噬菌体文库淘选。在这个过程中，淘选是在溶液中进行的，使用生物素/链霉亲和素珠分离系统，进行了三轮。从最后一轮中选择了 9 个与许多未知血浆蛋白紧密结合的单克隆噬菌体，其中 6 个直接用作交联基质，从血浆中纯化其相应的抗原。G5 和 E1 基质分离得到的蛋白质分别被鉴定为淀粉样蛋白和载脂蛋白 A-I 前体。结果表明，在非比较蛋白质组范围内的系统中，同时获得针对各种抗原的配体噬菌体是可行的，包括低丰度蛋白。
