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 micrograms pure lipoxygenase protein per 1 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 13S-hydroperoxy-9Z,13E-octadecadienoic acid) and arachidonic acid. With the latter substrate it exhibits a dual positional specificity (formation of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetranoic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-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.
Institute of Biochemistry, School of Medicine Charite, Humboldt University, Berlin, Germany.
H
H
Kühn
B J
BJ
Thiele
A
A
Ostareck-Lederer
H
H
Stender
H
H
Suzuki
T
T
Yoshimoto
S
S
Yamamoto
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000201:enzymology*
D011994:Recombinant Proteins; D004247:DNA; D001093:Arachidonate 15-Lipoxygenase