Expression and activity of recombinant AtCuAO1, AtCuAO2 and AtCuAO3 proteins. (A) Construct used to produce recombinant AtCuAOs in N. benthamiana: The PCR-amplified CuAO ORFs without the stop codon flanked by Gateway recombination cassette sequences were cloned in-frame with the TAP tag composed of: two copies of the protein A IgG binding domain, the 3C protease cleavage site, a six histidine stretch, and nine repeats of the myc epitope. The expression of TAP-tagged AtCuAOs is under the control of the duplicated 35S cauliflower mosaic virus promoter (2x35S), a tobacco mosaic virus translational enhancer (TMV-Ω), and a NOS terminator (NOSt). RB, T-DNA right border sequence; LB, T-DNA left border sequence. (B) Western blot analyses of recombinant AtCuAO proteins produced in N. benthamiana plants. Proteins present in total extract (TE) and in the eluate, obtained from IgG beads using 3C protease (+3C), were separated on an 8% SDS-PAGE gel and immunoblotted using the α-myc antibody. The highest MW band corresponds to the fusion protein containing the whole TAP-tag, while in the lower MW band the IgG binding domain has been cleaved. Molecular weight markers (kD) are shown on the right. (C) Amine oxidase activity of recombinant fusion proteins AtCuAO1-MYC9-His6 (AtCuAO1), AtCuAO2-MYC9-His6 (AtCuAO2) and AtCuAO3-MYC9-His6 (AtCuAO3) (1 μg) was determined as fluorescence corresponding to the amount of H2O2 released by oxidation of Put or Spd (1 mM), alone or pre-treated with the CuAO inhibitors aminoguanidine (AG, 0.5 mM) or 8-hydroxyquinoline (8HQL, 30 μM), using Amplex Red. Data are the means ± SE of three replicates. Bars represent standard errors.
Planas-Portell et al. BMC Plant Biology 2013 13:109 doi:10.1186/1471-2229-13-109