BACTERIAL EXPRESSION, PURIFICATION AND PARTIAL CHARACTERIZATION OF RECOMBINANT RABBIT RETICULOCYTE 15-LIPOXYGENASE

The recombinant rabbit reticulocyte 15-lipoxygenase has been expressed in E. coli with a yield of about 50-70 mug pure lipoxygenase protein per 1 of liquid culture. The enzyme has been purified to apparent homo geneity from the bacteria lysis supernatant by ammonium sulfate precip itation, and two consecutive steps of anion exchange chromatography on a Mono Q column. As the native enzyme the recombinant lipoxygenase ha s a molecular mass of 75 kDa, an isoelectric point of 5.5 and oxygenat es both linoleic acid (formation of 13S-hydroperoxy-9Z,13E-octadecadie noic acid) and arachidonic acid. With the latter substrate it exhibits a dual positional specificity (formation of 15S-hydroperoxy-5Z,8Z,Z,1 3E-eicosatetraenoic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetra enoic acid in a ratio of 12:1). Furthermore, the enzyme is capable of oxygenating biomembranes, as indicated by HPLC analysis of esterified oxygenated polyenoic fatty acids.