Scalable purification and characterization of the extracellular domain of human autotaxin from prokaryotic cells.

Abstract

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.

• Authors: Arayo Haga, Kazunori Hashimoto, Nobutada Tanaka, Kazuo T Nakamura, Yoshihiro Deyashiki
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 59 Issue 1 Pg. 9-17 (May 2008) ISSN: 1096-0279 [Electronic] United States
• PMID: 18249559 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Multienzyme Complexes
• Recombinant Proteins
• Phosphoric Diester Hydrolases
• Phosphodiesterase I
• alkylglycerophosphoethanolamine phosphodiesterase
• Pyrophosphatases

Topics

• Cell Line, Tumor
• Cell Movement (drug effects)
• Cell Proliferation (drug effects)
• Chromatography, Gel
• Cloning, Molecular
• Escherichia coli (metabolism)
• Fibrosarcoma
• Humans
• Multienzyme Complexes (isolation & purification, metabolism)
• Phosphodiesterase I (isolation & purification, metabolism)
• Phosphoric Diester Hydrolases (metabolism)
• Pyrophosphatases (isolation & purification, metabolism)
• Recombinant Proteins (isolation & purification, metabolism)