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

LARA, MARÍA VALERIA; MÜLLER, GABRIELA LETICIA; DRINCOVICH, MARÍA FABIANA; ANDREO, CARLOS SANTIAGO

Bariloche

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

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Previous studies indicated that different antibodies batches against the purified photosynthetic NADP-Malic enzyme (NADP-ME) from maize leaves cross-react with a 72 kDa protein., detected in different tissues of several plant species. A 72 kDa protein with low NADP-ME activity was also purified from several plant sources. Thus, this 72 kDa-protein was pointed out as a non-photosynthetic NADP_ME. Nevertheless, we recently found that the cDAN supossed to encode for this NADP-ME in maize roots, codifies in fact for a novel highly active 66 kDa NADP-ME. In this way, in order to identify the nature of the 72 kDa protein , we used the maize purified antibodies to perfom a scrrening of a cDNA tobacco library ; as Western blot analysis of otabcco leaf protein with these antibodies detected only a 72 kDa -signal. After the screenining, we obtained 8 independent clones, 4 of which were completly sequenced. Blasta analysisi of the sequence obtained indicated that it was 80 to 93% dientical to Hsp70 from different plant asouces. Prediction analysis of the sequence obtained (AY 253326) showed that codifies for a possilbe cytosolic 70,876 Da protien. The cDNA for this Hsp70 was connected in frame to a pET32 expression vecto to over express the protein in E. coli. The association between the recombinant ANDP-ME and Hsp70 proteins in vitro, suggest that such association can be the reason for the cross-reaction of the anti-purified maize ANDP-ME antibodies.