RECOMBINANT PRODUCTION OF THE ARABIDOPSIS MITOCHONDRIAL ENZYMES PROLINE DEHYDROGENASE AND P5C DEHYDROGENASE

FABRO, GEORGINA; DEZA-BORAU, GERMÁN; ALVAREZ, MARÍA ELENA

Buenos Aires

Congreso; Reunión conjunta de Sociedades de Biociencias; 2017

Diferentes sociedades de biociencias (SAIC, SAIB, SAB, etc)

The aim of our work is to determine the activity and oligomericstate of the Arabidopsis enzymes proline (Pro) dehydrogenase(ProDH) and P5C dehydrogenase (P5CDH) that participate fromPro catabolism in the matrix of plant mitochondria. We also needto evaluate if there is physical interaction between these proteinsand, if so, the protein domains involved. To address the above-mentionedissues in vitro we expressed the proteins as recombinantsin E. coli. Arabidopsis cDNA was used as template and adequateprimers designed to clone the full length open reading frame of thegenes ProDH1, ProDH2 y P5CDH without the mitochondrial transitpeptide, as well as mutant versions of ProDHs lacking the N-terminalor P5CDH without the C- terminal into the pMAL-p2p vector. Thisexpression plasmid allows N-terminal fusions to the Maltose BindingProtein (MBP), which, as we confirmed, increases the fraction ofsoluble fusion proteins obtained. It also contains a cleavage site forthe Prescission protease to detach the protein of interest from thetag. We used E. coli BL21DE to test different conditions to optimizethe soluble expression of the proteins (temperature, time and inducerconcentration ?IPTG-). Soluble fusion proteins were purified byaffinity chromatography with amylose resin and we could verify thateither tagged or untagged these were properly recognized by homemadepolyclonal antibodies developed in the laboratory against peptidesof ProDH and P5CDH. The activity of ProDH1 and ProDH2were determined by using spectrophotometry monitoring the reductionof the absorbance at 600nm of the acceptor DCPIP. Detailsabout experiments designed