Expression, purification, crystallization and preliminary X‐ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine

摘要: A high-resolution structure of the complex Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine mechanism substrate specificity enzyme and for rational design pharmacological modulators. Here, expression purification VchUPh crystallization are reported. Conditions were determined an automated Cartesian Dispensing System using The Classics, MbClass II Suites kits. Crystals VchUPh–thymidine (of dimensions ∼200–350 µm) grown by sitting-drop vapour-diffusion method ∼7 d at 291 K. solution consisted 1.5 µl (15 mg ml−1), 1 µl 0.1 M reservoir [15%(w/v) PEG 4000, 0.2 M MgCl2.6H2O Tris–HCl pH 8.5]. crystals diffracted 2.12 A resolution belonged space group P21 (No. 4), unit-cell parameters a = 91.80, b = 95.91, c 91.89 A, β 119.96°. Matthews coefficient was calculated as 2.18 A3 Da−1; corresponding solvent content 43.74%.