Department of Life Sciences, University of Trieste, Trieste, Italy.
Nucleic Acids Res. 2010 May;38(9):e110. doi: 10.1093/nar/gkq052. Epub 2010 Feb 9.
We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.
我们开发了一种高通量的蛋白质表达和相互作用分析平台，它结合了 cDNA 噬菌体展示文库选择和使用 454 平台的大规模基因测序。从不同组织的 mRNA 中创建了一个开放阅读框 (ORF) 片段的噬菌体展示文库。该文库用于研究酶转谷氨酰胺酶 2 (TG2) 的相互作用网络，TG2 是一种多功能酶，参与细胞生长、分化和凋亡的调节，与许多不同的病理有关。用 TG2 进行两轮淘选后，我们使用 454 测序法检测了所选噬菌体群体中 ORF 的频率。对超过 120000 个序列进行排序和分析，使我们能够识别出几个潜在的相互作用蛋白，随后在功能测定中得到了证实。在所鉴定的克隆中，有三个以前被描述为相互作用蛋白（纤连蛋白、SMOC1 和 GSTO2），而其他所有蛋白都是新的。与标准系统（如微量酶联免疫吸附测定）相比，这里描述的方法速度快得多，并且可以提供更多关于正在研究的相互作用的信息，从而更好地描述复杂系统。例如，在纤连蛋白的情况下，能够鉴定出参与相互作用的特定结构域。
