title: Purification and crystallization of yeast Ent1 ENTH domain.
abstract: Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS-PAGE analysis of dissolved crystals.
authors: Department of Biochemistry and Molecular Biology, The Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors: Neta
authors: N
authors: Tanner
authors: Gali
authors: G
authors: Prag
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: C121319:ENT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins