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首页> 外文期刊>Journal of Molecular Biology >SELECTIVELY-INFECTIVE PHAGE (SIP) - A MECHANISTIC DISSECTION OF A NOVEL IN VIVO SELECTION FOR PROTEIN-LIGAND INTERACTIONS
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SELECTIVELY-INFECTIVE PHAGE (SIP) - A MECHANISTIC DISSECTION OF A NOVEL IN VIVO SELECTION FOR PROTEIN-LIGAND INTERACTIONS

机译：选择性感染噬菌体（SIP）-体内选择蛋白质-配体相互作用的新机制

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Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a Ligand-binding protein, and (2) an ''adapter'' molecule in which the ligand is Linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions. (C) 1997 Academic Press Limited. [References: 44]
机译：选择性感染噬菌体（SIP）是一种用于体内选择相互作用的蛋白质-配体对的新颖方法。它由两个部分组成：（1）通过用配体结合蛋白替换其在GeneIII蛋白（gIIIp）的N端结构域而变成非感染性的噬菌体颗粒，和（2）“适配子”分子，其中配体与噬菌体颗粒缺失的gIIIp的N末端结构域相连。当展示的蛋白质与配体结合，从而使缺失的gIIIp的N末端结构域与噬菌体颗粒结合时，感染性得以恢复。因此，噬菌体繁殖严格取决于蛋白质-配体相互作用。我们已经表明，将β-内酰胺酶插入gIIIp的不同位置，模仿了蛋白质-配体对的插入，可导致高度感染性的噬菌体颗粒。缺少第一个N末端结构域的任何噬菌体根本没有感染性。相反，仅缺乏第二个N-末端结构域的那些细胞显示低的感染性，而与受体细胞上是否存在F-菌毛无关，这可以通过添加钙来增强。将抗荧光素scFv抗体及其抗原荧光素作为SIP实验的蛋白质-配体模型系统进行了检查。通过荧光素与纯化的N末端域化学偶联合成的衔接子分子与非感染性抗荧光素scFv展示噬菌体混合。感染事件严格取决于与N末端域偶联的荧光素，并强烈依赖于衔接子浓度。与仅一个抗原独立事件相比，从10（10）个输入噬菌体可以获得多达10（6）个抗原特异性事件。由于无需分离粘合剂和非粘合剂，因此SIP有望作为一种选择高亲和力相互作用的快速方法。 （C）1997 Academic Press Limited。 [参考：44]

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