M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins.
M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins.
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DOI：
10.1021/bc300130z
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发表时间：
2012-07-18
影响因子：
4.7
通讯作者：
Guimaraes CP
中科院分区：
文献类型：
--
作者：
Hess GT;Cragnolini JJ;Popp MW;Allen MA;Dougan SK;Spooner E;Ploegh HL;Belcher AM;Guimaraes CP
我们利用细菌分选酶将各种部分连接到M13噬菌体的衣壳蛋白。我们表明，pIII、pIX和pVIII可以用小分子（例如，荧光团，生物素）到正确折叠的蛋白质（例如，GFP，抗体，链霉亲和素），并与产量超过任何报道的使用噬菌体展示技术。一个恰当的例子是pVIII的修饰。虽然噬菌体载体将插入pVIII的大小限制为几个氨基酸，但噬菌粒系统限制了实际展示在M13表面的拷贝数。使用基于分选酶的反应，实现了GFP在pVIII上的展示效率的100倍增加。利用正交分选酶，我们可以同时靶向同一噬菌体颗粒中的两种不同的衣壳蛋白，并保持良好的标记特异性。正如这项工作所证明的那样，这是一种简单有效的方法，可以创建各种结构，从而扩大了M13在材料科学应用中的用途，并作为生物工具。
We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.