Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein.
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.
Hausmann
Jens
J
Division of Biochemistry B8, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Christodoulou
Evangelos
E
Kasiem
Mobien
M
De Marco
Valeria
V
van Meeteren
Laurens A
LA
Moolenaar
Wouter H
WH
Axford
Danny
D
Owen
Robin L
RL
Evans
Gwyndaf
G
Perrakis
Anastassis
A
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
D006023:Glycoproteins; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D000099189:Lysophospholipase D
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