Expression, purification, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris.
A cDNA coding for porcine leukocyte 12-lipoxygenase was expressed intracellularly in the methylotrophic yeast Pichia pastoris under the regulatory control of the alcohol oxidase promoter. The recombinant 12-lipoxygenase contained in the yeast cell lysate was soluble, displayed the catalytic properties of the native enzyme, and was recognized by antibodies prepared against native 12-lipoxygenase derived from porcine leukocytes. The catalytically active enzyme of the 100,000 x g supernatant obtained from the yeast lysate was readily purified by immunoaffinity chromatography to near homogeneity. Porcine leukocyte 12-lipoxygenase is the first arachidonic acid oxygenase to be expressed in yeast, an easy, inexpensive, and rapid method of expressing native and site-directed mutants of recombinant proteins.
Reddy
R G
RG
A.B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Yoshimoto
T
T
Yamamoto
S
S
Marnett
L J
LJ
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007962:Leukocytes / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001092:Arachidonate 12-Lipoxygenase