Scalable purification and characterization of the extracellular domain of human autotaxin from prokaryotic cells.
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.
Haga
Arayo
A
Research Institute for Health and Environmental Science, Gifu Prefectural Government, 1-1, Naka-Fudougaoka, Kakamigahara 504-0838, Japan. haga-arayo@pref.gifu.lg.jp
Hashimoto
Kazunori
K
Tanaka
Nobutada
N
Nakamura
Kazuo T
KT
Deyashiki
Yoshihiro
Y
D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005354:Fibrosarcoma; D006801:Humans; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D043264:Phosphodiesterase I / Q000302:isolation & purification* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D000099189:Lysophospholipase D
D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D000099189:Lysophospholipase D