Sceening a tobacco cDNA library with purified maize NADP-ME antibodies and expression of the product obtained in E. coli

MARÍA V. LARA; GABRIELA L. MÜLLER; MARÍA F. DRINCOVICH; CARLOS S. ANDREO

Bariloche

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2003

Previous studies indicated that different antibodies´ batches againstthe purified photosynthetic NADP-malic enzyme (NADP-ME)from maize leaves cross-react with a 72 kDa protein, detected indifferent tissues of several plant species. A 72 kDa protein withlow NADP-ME activity was also purified from several plantsources. Thus, this 72 kDa-protein was pointed out as a nonphotosyntheticNADP-ME. Nevertheless, we recently found thatthe cDNA supposed to encode for this NADP-ME in maize roots,codifies in fact for a novel highly active 66 kDa NADP-ME. Inthis way, in order to identify the nature of the 72 kDa protein, weused the maize purified NADP-ME antibodies to perform ascreening of a cDNA tobacco library; as Western blot analysis oftobacco leaf protein with these antibodies detected only a 72 kDasignal.After the screening, we obtained 8 independent clones, 4of which were completely sequenced. Blast analysis of thesequence obtained indicated that it was 80 to 93% identical toHsp70 from different plant sources. Prediction analysis of thesequence obtained (AY253326) showed that codifies for a possiblecytosolic 70,876 Da protein. The cDNA for this Hsp70 wasconnected in frame to a pET32 expression vector to over-expressthe protein in E. coli. The association between the recombinantNADP-ME and Hsp70 proteins in vitro, suggest that suchassociation can be the reason for the cross-reaction of the antipurifiedmaize NADP-ME antibodies.