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KMID : 0624620100430080541
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BMB Reports
2010 Volume.43 No. 8 p.541 ~ p.546
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Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells
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Song Yuanda
Dilger Emily
Bell Jessica
Barton William A
Fang Xianjun
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Abstract
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We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.
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KEYWORD
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Autotaxin, Cell motility, Cytokines, Lysophosphatidic acid, Lysophospholipase D, Mammalian cells
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