Slootweg Erik J, Keller Hans J H G, Hink Mark A, Borst Jan Willem, Bakker Jaap, Schots Arjen
Laboratory of Molecular Recognition and Antibody Technology, Wageningen University Binnenhaven 5, 6709 PD, Wageningen, The Netherlands.
Nucleic Acids Res. 2006;34(20):e137. doi: 10.1093/nar/gkl600. Epub 2006 Oct 13.
Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3' end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.
裂解性噬菌体形成了一个用于展示大型cDNA文库的强大平台，并提供了筛选与几乎任何底物相互作用的可能性。为了通过荧光显微镜直接观察这些相互作用，我们利用噬菌体的灵活性来整合其衣壳蛋白的修饰版本，构建了荧光T7噬菌体。通过应用翻译移码序列，构建了辅助质粒，该质粒表达固定比例的野生型衣壳蛋白（gp10）和与增强型黄色荧光蛋白（EYFP）融合的衣壳蛋白。移码序列插入在衣壳基因的3'末端与编码EYFP的序列之间。荧光融合蛋白仅在核糖体在翻译过程中使阅读框发生-1移位时形成。使用标准荧光显微镜，我们可以灵敏地监测在荧光T7噬菌体上展示的cDNA文库中特异性结合物的富集情况。本文还讨论了荧光展示噬菌体在快速发展的单分子检测和分选技术领域中的前景。
