Recombinant Expression of a Truncated Arabidopsis AX4-like Protein and Molecular Characterization of its Enzymatic Activities

Abstract

Adenylate cyclases (ACs) are part of a group of integral membrane proteins that consist of six transmembrane segments and have two emerging catalytic domains. They are known for their role in catalyzing the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP). This resultant cAMP molecule has been implicated in a number of plant processes for example signal transduction and responses to various environmental factors such as nutrient shortage and pest attacks. The premise of this research study was therefore laid upon the study of these ACs and their potential in vivo enzymatic activities. Based on the conserved and functionally assigned residues in the catalytic centre of annotated and experimentally confirmed nucleotide cyclases, various potential AC candidates in the Arabidopsis thaliana genome were annotated. Therefore in an attempt to test and determine whether this putative protein candidate has any functional AC activity, total mRNA of the 4-6 weeks old Arabidopsis thaliana plants was extracted and used as a template for the complementary synthesis and amplification of a 714 bp AC-like gene fragment via a specialized Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) system. The amplified fragment was then cloned into a pTrcHis2-TOPO expression vector and the resultant recombinant expression vector eventually transformed into chemically competent E.cloni EXPRESSBL21 (DE3) pLysS expression host cells. Positive clones were determined by confirmatory PCR and further validated by nucleotide-specific sequencing. The 20.0 kDa C-terminus His-tagged recombinant AC-like protein was the over-expressed following an induction with isopropyl-β-D-1-thiogalactopyranoside (1 mM, IPTG) and purified over a nickel nitrilotriacetic acid (NiNTA) affinity matrix system. The endogenous and in vitro AC activities of the resultant recombinant AC-like protein were then tested via a cAMP-linked enzyme immunoassaying system while its inherent in vivo AC activity was also concurrently tested via a complementation testing system using the cyaA SP850 mutant Escherichia coli cells. Results from these three independent assays collectively indicated that the AC-like protein encoded for At1g73980 gene from A.thaliana possesses the endogenous, in vitro and in vivo AC activities, and thus unequivocally confirming it as a bona fide higher plant AC molecule with a possible cAMP-mediated signalling system. One such candidate molecule in the form of an AX4-like protein was hereby cloned and partially expressed followed by its intensive functional characterization. Results from all these experimental approaches, practically showed that this putative AX4-like protein has some inherent adenylate cyclase