School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
Biotechnol J. 2014 Mar;9(3):355-61. doi: 10.1002/biot.201300462. Epub 2014 Jan 29.
The ability of Cre recombinase to excise genetic material has been used extensively for genome engineering in prokaryotic and eukaryotic cells. Recently, split-Cre fragments have been described that advance control of recombinase activity in mammalian cells. However, whether these fragments can be utilized for monitoring protein-protein interactions has not been reported. In this work, we developed a protein-fragment complementation assay (PCA) based on split-Cre for monitoring and engineering pairwise protein interactions in living Escherichia coli cells. This required creation of a dual-fluorescent reporter plasmid that permits visualization of reconstituted Cre recombinase activity by switching from red to green in the presence of an interacting protein pair. The resulting split-Cre PCA faithfully links cell fluorescence with differences in binding affinity, thereby allowing the facile isolation of high-affinity binders based on phenotype. Given the resolution of its activity and sensitivity to interactions, our system may prove a viable option for poorly expressed or weakly interacting protein pairs that evade detection in other PCA formats. Based on these findings, we anticipate that our split-Cre PCA will become a highly complementary and useful new addition to the protein-protein interaction toolbox.
Cre 重组酶切除遗传物质的能力已被广泛用于原核和真核细胞的基因组工程。最近，已经描述了分裂 Cre 片段，可用于哺乳动物细胞中重组酶活性的控制。然而，这些片段是否可用于监测蛋白质-蛋白质相互作用尚未报道。在这项工作中，我们开发了一种基于分裂 Cre 的蛋白质片段互补测定法 (PCA)，用于监测和工程化活大肠杆菌细胞中两两蛋白质相互作用。这需要创建一个双荧光报告质粒，该质粒允许在存在相互作用的蛋白质对时通过从红色切换到绿色来可视化重新组装的 Cre 重组酶活性。所得的分裂 Cre PCA 将细胞荧光与结合亲和力的差异精确地联系起来，从而可以根据表型轻松分离高亲和力结合物。鉴于其活性的分辨率及其对相互作用的敏感性，我们的系统可能为其他 PCA 格式中难以检测的表达量低或相互作用弱的蛋白质对提供可行的选择。基于这些发现，我们预计我们的分裂 Cre PCA 将成为蛋白质-蛋白质相互作用工具包的一个高度互补和有用的新补充。
