Development of phage surface engineering for green biotechnology

我们设计了一个噬菌体T2系统来展示T2噬菌体头蛋白小外衣壳（SOC） N端融合的蛋白。为了便于嵌合噬菌体的选择，构建了在soc基因上游编码β-半乳糖苷酶基因（βgal）的T2噬菌体。这种噬菌体被命名为2β gal，在含有XGal的琼脂板上产生蓝色斑块。随后，构建了一个编码soc上游目标蛋白的质粒，并将其用于转化大肠杆菌B^E细胞。用2 βgal感染转化后的细胞，噬菌体DNA与质粒同源重组产生嵌合噬菌体，由于βgal基因的切除，嵌合噬菌体产生透明斑块。以芽孢杆菌菌株K17 （ChoK）壳聚糖酶为模型靶蛋白，该酶由453个氨基酸组成。产生ChoK的重组T2噬菌体命名为T2ChoK。T2ChoK由T2βGal以约0.1%的重组频率产生。另一方面，野生型T2产生的T2IβGal值为0.001 %。该系统使我们能够快速、准确地选择重组噬菌体。计算每个噬菌体的ChoK分子数为14.7。估计了嵌合噬菌体的潜伏期和爆发大小。

We designed a bacteriophage T2 system to display proteins fused at the N terminus of the head protein small outer capsid (SOC) of T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the β-galactosidase gene (βgal) upstream of the soc gene was constructed. The phage, named T2βGal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E.coli B^E cells. Transformed cells were infected with T2βGal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage which produced transparent plaques due to the excision of the βgal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage which produced ChoK was named T2ChoK. T2ChoK was produced from T2βGal at a recombination frequency of about 0.1 %. On the other hand, the value for T2IβGal produced from wild type T2 was 0.001 %. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period, and burst size were estimated for chimeric phages.