title: Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein.
abstract: Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an approximately 100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 A resolution and allowed the collection of complete diffraction data to about 2.6 A resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.
authors.affiliation: Division of Biochemistry B8, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Jens
authors.initials: J
authors.lastname: Hausmann
authors.forename: Evangelos
authors.initials: E
authors.lastname: Christodoulou
authors.forename: Mobien
authors.initials: M
authors.lastname: Kasiem
authors.forename: Valeria
authors.initials: V
authors.lastname: De Marco
authors.forename: Laurens A
authors.initials: LA
authors.lastname: van Meeteren
authors.forename: Wouter H
authors.initials: WH
authors.lastname: Moolenaar
authors.forename: Danny
authors.initials: D
authors.lastname: Axford
authors.forename: Robin L
authors.initials: RL
authors.lastname: Owen
authors.forename: Gwyndaf
authors.initials: G
authors.lastname: Evans
authors.forename: Anastassis
authors.initials: A
authors.lastname: Perrakis
mesh_terms: D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D000099189:Lysophospholipase D
chemical_list: D006023:Glycoproteins; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D000099189:Lysophospholipase D
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