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Biochem Biophys Res Commun. 1994 Nov 30;205(1):381-8.

Expression, purification, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris.

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  • 1A.B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.

Abstract

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.

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