Recombinant Expression of Arabidopsis thaliana SNRK1 Signalling Cascade

ROJAS, BE; PIATTONI, CV; IGLESIAS, AA

Mar del Plata

Congreso; LI Reunión Anual de SAIB; 2015

SAIB

The SnRK1 signalling cascade is involved in cell energy homeostasis maintenance in plants. The system is activated by starvation conditions and triggers changes in gene expression and enzyme activity by phosphorylation, thus regulating carbon/nitrogen metabolism. In Arabidopsis, SnRK1 is composed of 9 genes and its mode of action and regulation remains inconclusive. We developed a recombinant strategy to express (co-express) in E. coli proteins from the Arabidopsis SnRK1 signalling cascade: an SnRK1 catalytic subunit, regulatory subunits ( and ) and the Geminivirus Rep-Interacting Kinase 1 (GRIK1, At3g45240). The expression and purification strategy was optimized to obtain a recombinant His6-tagged GRIK1 in (auto)phosphorylated form. Besides when the latter was co-expressed with the SnRK1 2 both proteins resulted phosphorylated. Using a spectrophotometric kinase assay coupled to PyRKase/LDHase enzymes, we determined that the recombinant His6-tagged GRIK1 was active an able to transphosphorylate and activate the SnRK1 2 subunit. Results indicate that co-expression strategies would provideadequate molecular tools to produce different SnRK1 kinases. The characterization of the target specificity and the kinetic/regulatory properties of these recombinant SnRK1 kinases would critically contribute to better understand the regulation of carbon and nitrogen metabolism in plant cells.