Abstract
The recombinant rabbit reticulocyte 15-lipoxygenase has been expressed in E coli with a yield of about 50–70 βg pure lipoxygenase protein per l of liquid culture. The enzyme has been purified to apparent homogeneity from the bacteria lysis supernatant by ammonium sulfate precipitation, and two consecutive steps of anion exchange chromatography on a Mono Q column. As the native enzyme the recombinant lipoxygenase has a molecular mass of 75 kDa, an isoelectric point of 5.5 and oxygenates both linoleic acid (formation of 13 S-hydroperoxy-9 Z,13 E-octadecadienoic acid) and arachidonic acid. With the latter substrate it exhibits a dual positional specificity (formation of 15 S-hydroperoxy-5 Z,8 Z,11 Z,13 E-eicosatetraenoic acid and 12 S-hydroperoxy-5 Z,8 Z,10 E,14 Z-eicosatetraenoic 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.
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