Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.
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.
Song
Yuanda
Y
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.
Dilger
Emily
E
Bell
Jessica
J
Barton
William A
WA
Fang
Xianjun
X
D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D043264:Phosphodiesterase I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism; D000099189:Lysophospholipase D
D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D013917:Thrombin; D000099189:Lysophospholipase D
J Cell Sci. 2005 Jul 15;118(Pt 14):3081-9
15985467
Protein Expr Purif. 2008 May;59(1):9-17
18249559
Nat Struct Mol Biol. 2006 Jun;13(6):524-32
16732286
Genes Cells. 2006 Feb;11(2):133-42
16436050
J Biol Chem. 2006 Sep 1;281(35):25822-30
16829511
Mol Cell Biol. 2006 Jul;26(13):5015-22
16782887
Cancer Cell. 2009 Jun 2;15(6):539-50
19477432
J Biol Chem. 2006 Sep 8;281(36):26081-8
16837466
J Pharmacol Toxicol Methods. 2005 May-Jun;51(3):187-200
15862464
Mol Biol Cell. 2008 Dec;19(12):5435-45
18843048
J Biol Chem. 2005 Jun 3;280(22):21155-61
15769751
Biochim Biophys Acta. 2007 Jan;1771(1):93-102
17208043
J Biol Chem. 2002 Oct 18;277(42):39436-42
12176993
Crit Rev Biochem Mol Biol. 2000;35(6):393-432
11202013
J Biol Chem. 1992 Feb 5;267(4):2524-9
1733949
J Biol Chem. 2008 Mar 21;283(12):7776-89
18175805
J Cell Biol. 2002 Jul 22;158(2):227-33
12119361
J Biol Chem. 2004 Mar 5;279(10):9653-61
14670967
J Biol Chem. 2003 May 16;278(20):18162-9
12642576