Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39.
Acta Crystallogr Sect F Struct Biol Cryst Commun
; 64(Pt 11): 1063-5, 2008 Nov 01.
Article
em En
| MEDLINE
| ID: mdl-18997343
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Apirase
/
Antígenos CD
/
Estrutura Terciária de Proteína
Limite:
Animals
Idioma:
En
Revista:
Acta Crystallogr Sect F Struct Biol Cryst Commun
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos