Recombinant expression and functional characterization of a fusion F-box protein from A. thaliana

Abstract

Adenylate cyclases (A Cs) are enzymes that are capable of converting adenine-5'-triphosphate (ATP) to cyclic 3', 5'-adenosine monophosphate (cAMP). It has been found that cAMP has an important role in cell signalling and as a second messenger in animals, plants and lower eukaryotes. Cyclic 3', 5'-adenosine monophosphate can affects many different physiological and biochemical processes such as the activities of kinases. However and up to date, the only annotated and experimentally confirmed AC in higher plants is the Zea mays pollen capable of generating cAMP and is involved in the growth of polarized pollen tubes. Recently, an Fbox protein from Arabidopsis thaliana has been bioinformatically annotated as a possible higher plant AC because of its possession of a putative adenylate cyclase catalytic motif in its structural domain. r n this study therefore, the main aim was to test and determine if the putative AC containing segment of the F-box gene has any functional AC activity and if so, to further explore if it has any physiological roles in plant cell signaling and transduction systems. Therefore, in order to attempt this aspect, putative AC containing segment of the Fbox gene was cloned into a prokaryotic expression vector (pGex-6p2) and expressed in E. coli BL21 (DE3) pLysS cells. In order to demonstrate the biological functionality of the Fbox's adenylate cyclase catalytic centre, the recombinant protein was tested for its ability to generate cAMP endogenously, in vitro and in vivo. Results from these three assays all indicated that the recombinant F-box-AC does possess functional adenylate cyclase activity.