Recombinant expression and functional characterization of a putative pentatrico-peptide protein from Arabidopsis thaliana
Dikobe, Bridget Tshegofatso
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Food security appears to be heavily dependent on the development of crop plants with increased resistance to both biotic and abiotic stresses such as pathogen infections and droughts respectively. Plant biotechnology has focused strongly on protein molecules that systemically affect homeostasis in plants and, one such possible candidate molecule is the pentatricopeptide protein, whose gene (PPR, Atlg62590) has recently been bioinformatically identified from the Arabidopsis genome and it harbours an adenylate cyclase (AC) catalytic motif. Adenylate cyclases (ACs) are enzymes that are capable of converting adenine triphosphate (ATP) to cyclic adenosine monophosphate ( cAMP) whose purpose is to function as a second messenger molecule in various phys iological and biochemical cell signalling systems. The PPR protein family has previously been experimentally shown to have roles in RNA processing and the restoration of cytoplasmic male sterility. However, to date, there has been no study to characterise if the PPR protein encoded by Atl g62590 has also AC activity. Therefore the aim of this study was to confirm if the PPR protein encoded by At ! g62590 has any AC activity and if so, to further explore if it has any physiological roles in plant cell signalling systems. In order to attempt this aspect, the putative AC containing PPR gene was cloned in a prokaryotic expression vector (pCR®T7 TOPO®-NT) and expressed in E. coli BL2 l (DE3) pLysS cells. In order to demonstrate the biological functionality of the PPR'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 PPR-AC does possess adenylate cyclase activity.