BMB Reports

생화학분자생물학회 (Korean Society for Biochemistry and Molecular Biology)
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Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells

  • Song, Yuanda (Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine) ;
  • Dilger, Emily (Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine) ;
  • Bell, Jessica (Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine) ;
  • Barton, William A. (Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine) ;
  • Fang, Xianjun (Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine)
  • 투고 : 2010.05.24
  • 심사 : 2010.07.15
  • 발행 : 2010.08.31
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초록

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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참고문헌

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  3. Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening vol.19, pp.7, 2014, https://doi.org/10.1177/1087057114520975