Recombinant Expression of a Putative F-box Protein from Arabidopsis thaliana and Testing for its Possible Adenylate Cyclase Activity

Abstract

Cyclic 3',5' adenosine monophosphate (cAMP) is a product of ATP hydrolysis catalyzed by the enzymes adenylate cyclases (ACs). These two groups of molecules (cAMP and ACs) have both been experimentally confirmed as key components and second messengers in the cell signaling cascades of both animals and lower eukaryotes. They play a vital role in linking environmental stimuli to physiological response in living cells. Although this information is widely documented in animals and lower eukaryotes, in plants both the presence and biological role of ACs are still very obscure and elusive. In this study, our work focused on an Arabidopsis thaliana F-box protein (coded for by the At4g39756 gene), which recently has been bioinfonnatically annotated as an AC candidate and at the same time, previously been implicated in stress responses, for assessment of its possible adenylate cyclase activity. In order to assess for this possible AC activity, we extracted total mRNA from 8-weeks old Arabidopsis thaliana leaf material and used this mRNA as a template for the amplification of the At4g39756 gene fragment bearing the putative AC catalytic center in the F-box domain using RT-PCR technique. The amplified fragment was then cloned into a pCRT7/NT-TOPO vector and the resultant recombinant construct (pCRT7/NT-TOPO:F-box) then subsequently transformed into the expression host, Escherichia coli BL21 (DE3) Star pLysS cells. Positive transformants were then cultured in a double strength Yeast-Trytone Soy broth and induced with I Mm IPTG at an OD6oo 0.5 to express the recombinant F-box protein. The expressed recombinant protein was found to be in its insoluble and non-native form and was therefore, first purified on a Ni-NTA column under denaturing conditions followed by its re-naturing into its native and soluble form via a linear gradient on the same colwnn. The resultant protein product was then tested for its possible endogenous, in vitro and in vivo AC activities. The endogenous and in-vitro AC activities were tested and determined by a cAMP-specific enzyme immunoassaying system while the in-vivo activity was tested and determined via a complementation testing. Results from all the three tests showed that this putative F-box protein has some inherent endogenous, in-vitro and in-vivo adenylate cyclase activities and therefore confirming it as a higher plant adenylate cyclase with a possible cAMP-mediated signaling system.