title: Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39.
abstract: 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 P3(2), with unit-cell parameters a = b = 118.1, c = 81.6 A 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 A 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 P3(2) lattice and rigid-body refined and position-minimized with PHENIX.
authors.affiliation: Department of Chemical and Screening Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA. xzhong@wyeth.com
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhong
authors.forename: Madhavan
authors.initials: M
authors.lastname: Buddha
authors.forename: Guido
authors.initials: G
authors.lastname: Guidotti
authors.forename: Ron
authors.initials: R
authors.lastname: Kriz
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.forename: Lidia
authors.initials: L
authors.lastname: Mosyak
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D015703:Antigens, CD; D011993:Recombinant Fusion Proteins; D001081:Apyrase; C067869:CD39 antigen