We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011 pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.

中文翻译：

---

M13噬菌体衣壳蛋白IX的高效亲和标记技术，用于将蛋白III展示的寡肽探针固定在非生物平台上。

我们开发了一种遗传方法，可以有效地向人群中M13丝状噬菌体的每个蛋白IX（pIX）拷贝添加亲和标签。可以将亲和标记的噬菌体固定在均匀的单层表面上，以定位pIII展示的肽或蛋白质，以使其与配体最佳相互作用。标记包括两个主要步骤。首先，通过与gIX :: aacCI插入等位基因的基因重组，在大肠杆菌中突变了M13噬菌体的基因IX（gIX）。其次，将共同产生亲和标签的pIX和天然pVIII的质粒转化到携带有缺陷M13 gIX的菌株中。这种遗传互补允许形成带有噬菌体颗粒的感染性噬菌体颗粒，该噬菌体颗粒带有亲和标签的pIX的完整补体（每个病毒体5个拷贝）。为了证明我们方法的有效性，我们用链球菌标签II标记了M13衍生噬菌体M13KE。为了用Strep-tag II标记pIX，从pASG-IBA4克隆并表达了pIX和pVIII的噬菌体基因，所述pASG-IBA4在四环素启动子/操作系统的控制下包含大肠杆菌OmpA信号序列和Strep-Tag II。当用10 ng / ml脱水四环素诱导Strep-Tag II-pIX-pVIII融合蛋白时，我们实现了3×1011 pfu / ml的最大噬菌体产量。亲和标记噬菌体探针的完整过程不到5天，可用于标记任何M13或fd pIII展示的寡肽探针，以改善其性能。大肠杆菌OmpA信号序列和Strep-Tag II在四环素启动子/操作系统的控制下。当用10 ng / ml脱水四环素诱导Strep-Tag II-pIX-pVIII融合蛋白时，我们实现了3×1011 pfu / ml的最大噬菌体产量。亲和标记噬菌体探针的完整过程不到5天，可用于标记任何M13或fd pIII展示的寡肽探针，以改善其性能。大肠杆菌OmpA信号序列和Strep-Tag II在四环素启动子/操作系统的控制下。当用10 ng / ml脱水四环素诱导Strep-Tag II-pIX-pVIII融合蛋白时，我们实现了3×1011 pfu / ml的最大噬菌体产量。亲和标记噬菌体探针的完整过程不到5天，可用于标记任何M13或fd pIII展示的寡肽探针，以改善其性能。