Open AccessMammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein
Author(s) -
Hausmann Jens,
Christodoulou Evangelos,
Kasiem Mobien,
De Marco Valeria,
Van Meeteren Laurens A.,
Moolenaar Wouter H.,
Axford Danny,
Owen Robin L.,
Evans Gwyndaf,
Perrakis Anastassis
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309110032938
Subject(s) - autotaxin , lysophosphatidic acid , lysophosphatidylcholine , lysophospholipase , glycoprotein , biochemistry , hek 293 cells , phosphodiesterase , transfection , recombinant dna , chemistry , biology , enzyme , microbiology and biotechnology , phospholipase , phospholipid , receptor , phosphatidylcholine , gene , membrane
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.