Characterisation of TomLOXF. A. The detection of his-tagged proteins was realized on Western blot with an anti-His-tag antibody (1 and 2) and we evaluated the purity of the protein through SDS-PAGE and Coomassie blue staining (3). 1: Page Ruler Plus Prestained Protein Ladder (Fermentas), 1: total proteins extracted from TomLOXF-expressing E. coli, 2 and 3: partially-purified protein extracted from TomLOXF-expressing clone by nickel affinity chromatography.B. LOX activity was evaluated on partially-purified recombinant TomLOXF with linoleic (C18:2) and linolenic (C18:3) acids as substrate. Reaction was performed at pH 6.0, room temperature. C. Linolenic and linoleic acids were both incubated with extracts of E. coli expressing TomLOXF in oxygenated buffer. Produced hydroperoxides were separated by HPLC, and the profile of compounds absorbing at 234 nm was compared with the profile of pure 13-HPOT, 13-HPOD, 9-HPOT and 9-HPOD.