Open AccessExpression, purification and crystallization of the ecto‐enzymatic domain of rat E‐NTPDase1 CD39
Author(s) -
Zhong Xiaotian,
Buddha Madhavan,
Guidotti Guido,
Kriz Ron,
Somers Will,
Mosyak Lidia
Publication year - 2008
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309108032569
Subject(s) - crystallization , enzyme , domain (mathematical analysis) , chemistry , biochemistry , microbiology and biotechnology , biophysics , biology , organic chemistry , mathematics , mathematical analysis
CD39 is a prototype member of the ecto‐nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto‐enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction‐quality crystals of sCD39 were produced by the vapor‐diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P 3 2 , with unit‐cell parameters a = b = 118.1, c = 81.6 Å and with two sCD39 copies in the asymmetric unit. Several low‐ to medium‐resolution diffraction data sets were collected using an in‐house X‐ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular‐replacement search was performed against the complete 3.2 Å data set using a maximum‐likelihood molecular‐replacement method as implemented in Phaser . The initial model of the two sCD39 monomers was placed into the P 3 2 lattice and rigid‐body refined and position‐minimized with PHENIX.