Recombinant production of the Arabidopsis mitochondrial enzymes Proline dehydrogenase and P5C dehydrogenase

FABRO G; DEZA-BORAU G; ALVAREZ ME

Capital Federal, Buenos Aires

Congreso; LIII Reunión Anual SAIB; 2017

SAIB

The aim of our work is to determine the activity and oligomeric state of the Arabidopsis enzymes proline (Pro) dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) that participate from Pro catabolism in the matrix of plant mitochondria. We also need to evaluate if there is physical interaction between these proteins and, if so, the protein domains involved. To address the above-mentioned issues in vitro we expressed the proteins as recombinants in E. coli. Arabidopsis cDNA was used as template and adequate primers designed to clone the full length open reading frame of the genes ProDH1, ProDH2 y P5CDH without the mitochondrial transit peptide, as well as mutant versions of ProDHs lacking the N-terminal or P5CDH without the C- terminal into the pMAL-p2p vector. This expression plasmid allows N-terminal fusions to the Maltose Binding Protein (MBP), which, as we confirmed, increases the fraction of soluble fusion proteins obtained. It also contains a cleavage site for the Prescission protease to detach the protein of interest from the tag. We used E. coli BL21DE to test different conditions to optimize the soluble expression of the proteins (temperature, time and inducer concentration ?IPTG-). Soluble fusion proteins were purified by affinity chromatography with amylose resin and we could verify that either tagged or untagged these were properly recognized by homemade polyclonal antibodies developed in the laboratory against peptides of ProDH and P5CDH. The activity of ProDH1 and ProDH2 were determined by using spectrophotometry monitoring the reduction of the absorbance at 600nm of the acceptor DCPIP. Details about experiments designed to evaluate oligomerization state and interaction will be presented.