Novel assay system, based on the BiFC technique in Escherichia coli

Abstract

A novel assay system, based on the bi-molecular fluorescence complementation （BiFC） technique in Escherichia coli, was developed for detecting transient interactions such as those between kinases and their substrates. BiFC is an alternative technique for detecting protein-protein associations in live cells. This system detected, the interaction between OsMEK1 and its direct target, OsMAP1. By contrast, BiFC fluorescence was not observed when OsMAP2 or OsMAP3, which are not substrates of OsMEK1, were used as prey proteins. We also screened for interacting proteins of Ca²⁺-dependent protein kinase 8 （OsCPK8）, a regulator of plant immune responses, and identified three proteins as interacting molecules of OsCPK8. The interaction between OsCPK8 and two of these proteins （ARF-GEF and peptidyl prolyl isomerase） was confirmed in rice cells by means of BiFC technology. These results indicate that our new assay system has the potential to screen for protein kinase target molecules.