Haga Arayo, Hashimoto Kazunori, Tanaka Nobutada, Nakamura Kazuo T, Deyashiki Yoshihiro
Research Institute for Health and Environmental Science, Gifu Prefectural Government, 1-1, Naka-Fudougaoka, Kakamigahara 504-0838, Japan.
Protein Expr Purif. 2008 May;59(1):9-17. doi: 10.1016/j.pep.2007.12.008. Epub 2008 Jan 1.
Autotaxin (ATX) is an approximately 125kDa transmembrane protein known as a tumor progression factor based on its lysophospholipase D (lysoPLD) activity. There are many reports of the biological and biochemical properties of ATX, but crystallographic or structural studies have not been reported because a large-scale production process using prokaryotic cells has not been established. Here we report a bulk purification process and soluble expression of the recombinant human ATX (rhATX S48) from prokaryotic cells. The extracellular domain of human ATX cDNA was cloned into a pET101/D-TOPO vector and transformed to an Escherichia coliBL21 strain which was co-transformed with a pTF16 chaperone plasmid. The rhATX S48 was purified with chaperone and it was removed by Mg(2+)-ATP treatment. The final yield of purified rhATX S48 was approximately 3.5mg/l culture of recombinant strain. The rhATX S48 shows lysoPLD enzymatic activity and effectively stimulates the growth and motile activity of the human tumor cells as well as native ATX. This is a first report for scalable purification of the ATX molecule and the rhATX S48 should be a good tool for immunization of anti-ATX or crystallographic analysis of ATX.
自分泌运动因子（ATX）是一种分子量约为125kDa的跨膜蛋白，基于其溶血磷脂酶D（lysoPLD）活性，它被认为是一种肿瘤进展因子。关于ATX的生物学和生化特性已有许多报道，但由于尚未建立使用原核细胞的大规模生产工艺，因此尚未见晶体学或结构研究的报道。在此，我们报告了从原核细胞中大规模纯化重组人ATX（rhATX S48）及可溶性表达的方法。将人ATX cDNA的胞外域克隆到pET101/D-TOPO载体中，并转化到与pTF16伴侣质粒共转化的大肠杆菌BL21菌株中。rhATX S48通过伴侣蛋白进行纯化，并通过Mg(2+)-ATP处理将其去除。重组菌株培养物中纯化的rhATX S48的最终产量约为3.5mg/l。rhATX S48具有lysoPLD酶活性，并且与天然ATX一样，能有效刺激人肿瘤细胞的生长和运动活性。这是关于ATX分子可扩展纯化的首次报道，rhATX S48应该是抗ATX免疫或ATX晶体学分析的良好工具。
