Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.
BMB Rep. 2010 Aug;43(8):541-6. doi: 10.5483/bmbrep.2010.43.8.541.
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.
我们利用哺乳动物表达系统来纯化和表征自分泌酶（ATX）/溶血磷脂酶 D，这种酶存在于血液中，负责溶血磷脂酸的生物合成。编码氨基酸 29-915 的人 ATX cDNA 克隆在 CD5 的分泌信号下游。在羧基末端是一个凝血酶切割位点，其后是 IgG 的恒定区（Fc），以方便蛋白质纯化。ATX-Fc 融合蛋白在 HEK293 细胞中表达，并从稳定克隆的条件培养基中通过与 Protein A sepharose 的亲和层析分离，然后用凝血酶切割。未标记的 ATX 蛋白通过凝胶过滤层析进一步纯化至基本均一，产量约为每升培养基 5 毫克。纯化的 ATX 蛋白具有酶活性和生物学功能，为进一步研究这种重要酶的生物学和结构提供了有用的工具。
