Development of phage surface engineering for green biotechnology

我们设计了一个噬菌体T2展示系统，展示融合在T2噬菌体头部蛋白小外壳（SOC）N端的蛋白。为了便于选择嵌合噬菌体，构建了编码β-半乳糖苷酶基因（βgal）的T2噬菌体，所述β-半乳糖苷酶基因（βgal）位于β-半乳糖苷酶基因上游。命名为T2βGal的噬菌体在含有XGal的琼脂平板上产生蓝色噬斑。随后，构建了编码EST 3上游的靶蛋白的质粒，并用于转化大肠杆菌B^E细胞。用T2βGal感染转化细胞，噬菌体DNA与质粒之间的同源重组产生嵌合噬菌体，该嵌合噬菌体由于βgal基因的切除而产生透明噬斑。芽孢杆菌菌株K17（ChoK）的壳聚糖酶由453个氨基酸组成，被用作模型靶蛋白。将产生ChoK的重组T2噬菌体命名为T2 ChoK。从T2βGal以约0.1%的重组频率产生T2 ChoK。另一方面，由野生型T2产生的T2 I βGal的值为0.001%。这种新的系统使我们能够非常快速和准确地选择重组噬菌体。ChoK的分子数计算为每单个噬菌体14.7个。估计嵌合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.