Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39
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CitationZhong, Xiaotian, Madhavan Buddha, Guido Guidotti, Ron Kriz, Will Somers, and Lidia Mosyak. 2008. “Expression, Purification and Crystallization of the Ecto-Enzymatic Domain of Rat E-NTPDase1 CD39.” Acta Crystallographica Section F Structural Biology and Crystallization Communications 64 (11): 1063–65. https://doi.org/10.1107/s1744309108032569.
AbstractCD39 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 over-expressed in a glycosylation mutant CHO line, Lec. 22.214.171.124, 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 angstrom 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 angstrom 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.
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