Isoforma fotosintética de la enzima málica NADP-dependiente de maíz: clonado y complementación de una mutante de Escherichia coli por la expresión de una proteína de fusión

GERRARD WHEELER, M. C.; ANDREO, C. S.; DRINCOVICH, M. F.

Villa Carlos Paz, Argentina

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2001

SAIB

The photosynthetic isoform of NADP-malic enzyme (NADP-ME, E.C. 1.1.1.40), which catalyses the decarboxylation of L-malate to pyruvate and is located in bundle sheath chloroplasts in certain C4 plants such as maize, is an enzyme implicated in the photosynthetic process, yielding the CO2 to be fixed in bundle sheath chloroplasts by the Calvin cycle. Although several plant NADP-ME have been cloned and sequenced, none of them has been expressed in a bacterial system. In the present work, we describe the isolation of the full-length cDNA of NADP-ME from a maize leaf library constructed in UniZap XR. The entire coding region of the mature protein, along with a partial sequence of the transit peptide, was cloned in frame with the b-galactosidase gene. After expressing this fusion protein in E. coli, NADP-ME activity increased nearly 10-times from basal E. coli NADP-ME specific activity levels. This increase was accompanied by the appearance of a 69 kDa protein, which reacted with maize NADP-ME antibodies. A triple mutant of E. coli (strain EJ1321, pck-, dme- and tme-), which was unable to grow on minimal medium with C4 acids as sole carbon source, was complemented with maize NADP-ME protein expression. The entire coding region, along with a partial sequence of the chloroplastic transit peptide, was inserted into a pET32 expression vector to allow protein purification.