Den Blaauwen T, Wientjes F B, Kolk A H, Spratt B G, Nanninga N
Department of Molecular Cell Biology, University of Amsterdam, The Netherlands.
J Bacteriol. 1989 Mar;171(3):1394-401. doi: 10.1128/jb.171.3.1394-1401.1989.
We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.
我们制备了抗大肠杆菌青霉素结合蛋白1B（PBP 1B）的单克隆抗体，以研究该蛋白的膜拓扑结构、空间组织和酶活性。以PBP 1B作为免疫原产生的大多数抗体与羧基末端发生反应。为了获得识别其他表位的单克隆抗体，我们使用缺失免疫显性羧基末端65个氨基酸的PBP 1B作为免疫原。分离并鉴定了18种针对膜结合PBP 1B的单克隆抗体。用PBP 1B的各种截短形式确定了这些单克隆抗体识别的表位。我们可以区分位于分子不同部位的四个不同表位区域。有趣的是，尽管特意选择了针对氨基末端的单克隆抗体，但我们未能分离得到。这归因于其预测的极端亲水性和灵活性，这可能使氨基末端对蛋白水解降解非常敏感。在斑点印迹免疫结合试验中，所有抗体均与天然PBP 1B发生反应。一种单克隆抗体还识别完全十二烷基硫酸钠变性形式的PBP 1B。这表明所有其他单克隆抗体识别的是构象表位。这些特性使单克隆抗体成为进一步研究的合适工具。
