title: Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.
authors.forename: Yuanda
authors.initials: Y
authors.lastname: Song
authors.forename: Emily
authors.initials: E
authors.lastname: Dilger
authors.forename: Jessica
authors.initials: J
authors.lastname: Bell
authors.forename: William A
authors.initials: WA
authors.lastname: Barton
authors.forename: Xianjun
authors.initials: X
authors.lastname: Fang
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D043264:Phosphodiesterase I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism; D000099189:Lysophospholipase D
chemical_list: D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D013917:Thrombin; D000099189:Lysophospholipase D
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