Recombinant Expression and Functional Testing of Candidate Adenylate Cyclase Domains

Adenylate cyclases (ACs) are enzymes capable of converting adenosine-5′-triphosphate to cyclic 3′, 5′-�adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their product cAMP have firmly been established as important signalling molecules with important roles in several cellular signal transduction pathways. However, in higher plants, the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of generating cAMP. Recently a number of candidate AC-encoding genes in Arabidopsis thaliana have been proposed based on functionally assigned amino acids in the catalytic center of annotated and/or experimentally tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and recombinant expression of functional candidate AC domains using, as an example, the A. thaliana pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a complementation test, in vivo adenylate cyclase activity of candidate recombinant molecules can be prescreened and promising candidates can subsequently be further evaluated in an in vitro AC immunoassay.