Molecular cloning and expression of a phosphoenolpyruvate carboxykinase from Arabisopsis thaliana

LEADEN, L.; PODESTÁ, F.E.

Mar del Plata

Congreso; XLIII Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2007

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Phosphoenolpyruvate (PEP) carboxykinase (PEPCK) is an ubiquitous enzyme that catalises the ATP-dependent decarboxylation and phosphorylation of oxaloacetate to yield PEP, ADP and CO . Plant PEPCK has been assigned roles in gluconeogenesis and N assimilation. The study of the regulation of this key enzyme has been hampered by the extreme lability of its Nterminus (containing two phosphorylation sites of uncertain function in enzyme regulation) to proteolysis, that renders an active, yet truncated enzyme.With the goal of obtaining an active, intact form of PEPCK, we attempted the cloning and heterologous expression of a cDNA coding for a PEPCK from The source of cDNA was a fragment cloned in the PCMV-Sport6- PEPCK vector (INRA, CNRGV, Toulouse, Francia). This fragment was subcloned in pET-32a(+) with which strains BL21(DE3) and K12 JM109(DE3)pRIL were transformed, with poor expression or expression directed to the insoluble fraction being accomplished, respectively. Finally, expression in K12 JM109(DE3)pRILpKJE8 (using 30 µM IPTG) yielded a soluble form of the enzyme. Purification of the recombinant enzyme was accomplished with low yields, although activity could be measured. Further work aiming to enhance expression yield is on the way, paving the way for further studies on the impact of phosphorylation on enzyme activity and regulation.