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Mammalian cell expression purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein

DOI: 10.1107/S1744309110032938 DOI Help
PMID: 20823545 PMID Help

Authors: Jens Hausmann (Netherlands Cancer Institute) , Evangelos Christodoulou (Netherlands Cancer Institute) , Mobien Kasiem (Netherlands Cancer Institute) , Valeria De Marco (Netherlands Cancer Institute) , Laurens Van Meeteren (Netherlands Cancer Institute) , Wouter Moolenaar (Netherlands Cancer Institute) , Robin Owen (Diamond Light Source) , Gwyndaf Evans (Diamond Light Source) , Anastassis Perrakis (Netherlands Cancer Institute) , Danny Axford (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section F Structural Biology And Crystallization Communications , VOL 66 (9) , PAGES 1130-1135

State: Published (Approved)
Published: September 2010

Open Access Open Access

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 ~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 Å resolution and allowed the collection of complete diffraction data to about 2.6 Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.

Journal Keywords: Autotaxin; Crystallography

Subject Areas: Biology and Bio-materials, Medicine, Technique Development

Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: SLS

Added On: 01/09/2010 16:48

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