title: Expression, purification and crystallization of the human UL16-binding protein ULBP1.
abstract: UL16-binding proteins (ULBPs) are markers of cellular stress which are upregulated on the surface of virus-infected and tumor cells. Recognition of ULBP1 by the activating receptor NKG2D on the surface of cytotoxic natural killer (NK) and T cells promotes lysis of cells expressing ULBP1 and is an important mechanism of immune surveillance. We report a robust method for the generation of large quantities of crystal-grade recombinant ULBP1 protein. The extracellular portion of human ULBP1 was cloned into a T7 expression vector for expression in Escherichia coli. Unpaired cysteines in the sequence which are predicted not to be involved in the intramolecular disulfide bond formation were mutated to serine. ULBP1 was expressed in E. coli BL21 (DE3) pLysS cells as inclusion bodies. Purified inclusion bodies were solubilized by denaturation in guanidine, and refolded by slow dilution. The refolded protein was purified by size exclusion gel filtration and anion exchange chromatography. Furthermore, we have identified conditions optimal for the crystallization of this protein and have obtained initial diffraction data to 4.6Å from these crystals.
authors.affiliation: Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, UK.
authors.forename: Aleksandra A
authors.initials: AA
authors.lastname: Watson
authors.forename: Charita M
authors.initials: CM
authors.lastname: Christou
authors.forename: Christopher A
authors.initials: CA
authors.lastname: O'Callaghan
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D058851:GPI-Linked Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C423231:ULBP1 protein, human