A New And Quantitative Two-hybrid System To Detect Protein-protein Interactions In Escherichia Coli Based On Polyhydroxyalkanoates Synthesis Regulatory Proteins PhaR

Polyhydroxyalkanoate (PHA) synthesis regulatory protein PhaR contains a DNA binding domain (DBD) and a PHA granule binding domain (GBD),it anchors to the promoter region of PHA granule-associated protein (PhaP) to repress its expression. However,PhaR will bind to PHB granules and be released from the phaP promoter region when PHA granules are accumulated in vivo,initiating the expression of phaP gene. Based on this regulatory mechanism,a bacterial two-hybrid system to detect protein-protein interactions was developed: PhaR was separated into two parts: DNA binding domain DBD was used to fuse with the bait,PHA granule binding domain GBD with the prey,and phaP was replaced by a reporter gene, namely,β-galactosidase gene lacZ. However,GBD protein expressed in vivo formed inclusion bodies probably due to its high content of hydrophobic amino acid residues. Therefore,PhaP also with strong binding ability to PHB granules was employed to replace hydrophobic GBD. Three model interaction partners bFos,bJun and bATF2 were used to study the feasibility of the bacterial two-hybrid system compared with the controls lacking one or more essential elements of this system. Results showed that bFos,bJun and bATF2 bound tightly among themselves in pairs to allow strong expression ofβ-galactosidase in different expression levels. In contrast, very weakβ-galactosidase activity was detected in all other control groups. These results clearly demonstrated the feasibility of this system for studying protein interactions. Besides,according to significantly differentβ-galactosidase activity among all the pairwise combinations bFos:bJun,bJun:bJun and bJun:bATF2,we conclude this system can detect protein interactions quantitately.