A transferable heterogeneous two-hybrid system in <it>Escherichia coli </it>based on polyhydroxyalkanoates synthesis regulatory protein PhaR

• Main Authors: Chen Chong-Bo, Zhang Jing, Chen Jiong, Ma Ping, Wang Zhi-Hui, Chen Guo-Qiang
• Format: Article
• Language: English
• Published: BMC 2011-04-01
• Series: Microbial Cell Factories
• Online Access: http://www.microbialcellfactories.com/content/10/1/21

<p>Abstract</p> <p>Background</p> <p>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 <it>phaP </it>expression. However, PhaR will bind to PHB granules and be released from <it>phaP </it>promoter region when PHA granules are formed <it>in vivo</it>, initiating expression of <it>phaP </it>gene. Based on this regulatory mechanism, a bacterial two-hybrid system was developed: PhaR was separated into two parts: DBD was used to fuse with the bait, GBD with the prey, and <it>phaP </it>was replaced by a reporter gene <it>lacZ</it>. However, GBD protein expressed <it>in vivo </it>formed inclusion bodies. Thus, PhaP with strong binding ability to PHB granules was employed to replace GBD.</p> <p>Results</p> <p>Three model interaction partners bFos, bJun and bATF2 were used to study the feasibility of this 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 in pairs to allow strong expression of β-galactosidase in different expression levels. In contrast, very weak β-galactosidase activity was detected in all control groups.</p> <p>Conclusion</p> <p>β-Galactosidase activity level precisely correlated with the interaction force of tested protein pairs, and very weak β-galactosidase expression was detected throughout the control groups, which demonstrated the feasibility of this system for studying protein interactions.</p>