Molecular cloning, expresión and purification of orange fruit cytosolic pyruvate kinase

PEROTTI, V.; SKEJICH, A.; PODESTÁ, F.E.

Mar del Plata

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

SAIB

MOLECULAR CLONING, EXPRESSION AND PURIFICATION OF ORANGE FRUIT CYTOSOLIC PYRUVATE KINASE Perotti VE, SkejichAV, Podestá FE. CEFOBI-CONICET, Fac Cs Bioquímicas y Farmacéuticas, UNR, Suipacha 531, 2000 Rosario. E-mail: perotti@cefobi.gov.ar Pyruvate kinase (PK) catalyzes a reaction situated at a metabolic branchpoint in plant cytosolic carbon metabolism. In this energy conserving reaction ATP and pyruvate are generated from phosphoenolpyruvate and ADP, thus playing a pivotal role in the energy generation of plant cells. In addition, plant cytosolic PK (PK ) has been implicated in providing carbon skeletons for N assimilation and secondary metabolism. Pyruvate kinase cDNA was obtained by reverse transcription of mRNA isolated from orange fruit endocarp (C. sinensis var. Valencia late) followed by PCR using specific primers. The cDNAfragment was cloned in the expression vector pET-32a(+) as a 6xHis fusion protein. PKc was expressed in E. coli JM109(DE3)pRILpKJE8 cells and purified by one step Ni-chelating chromatography. The expressed protein was recognized by anti-His tail and anti B. napus-PKc antibodies. The heterologously expressed PK, after treatment with enterokinase, migrates as a single band of about 56 kDa as assessed by SDSPAGE. This work will allow the study at the molecular level of plant PK during development and in the mature fruit and perform kinetic and structural studies on the heterologously expressed