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

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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@article{Reddy1994ExpressionPA, title={Expression, purification, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris.}, author={Ram Gopal Reddy and Takanobu Yoshimoto and Satoshi Yamamoto and Lawrence J Marnett}, journal={Biochemical and biophysical research communications}, year={1994}, volume={205 1}, pages={381-8} }