title: Expression, refolding, purification, molecular characterization, crystallization, and preliminary X-ray analysis of the receptor binding domain of human B7-2.
abstract: The cell-mediated immune response involves a series of specific molecular interactions between cell surface molecules on T cells and antigen-presenting cells. Of particular importance for the regulation of T cell activity is the interaction of the B7 isoforms, B7-1 and B7-2, with the T cell surface costimulatory receptors, CD28 and CTLA-4. The binding of CD28 by B7-1/B7-2 results in an enhancement of T cell responses initiated by the interaction between a clonotypic T cell receptor and its specific, antigenic MHC-peptide complex, whereas the subsequent engagement of CTLA-4 by B7-1/B7-2 leads to a down-regulation of the response. Here we report the expression, refolding, purification, characterization, and crystallization of the receptor-binding domain of human B7-2. The receptor-binding domain of human B7-2 was overexpressed in Escherichia coli as inclusion bodies, solubilized in 6 M guanidine-hydrochloride, and then refolded in vitro by rapid dilution into a renaturing buffer. Refolded B7-2 was subsequently purified to homogeneity by anion-exchange chromatography. Gel-filtration chromatography and native PAGE analysis showed that the receptor-binding domain of B7-2 is exclusively monomeric in solution. Purified B7-2 binds tightly to bacterially expressed monomeric and disulfide-linked homodimeric human CTLA-4 as shown by gel-filtration chromatography and native PAGE. This suggests that glycosylation is not important for the proper folding of the receptor-binding domain of B7-2 nor for its binding to CTLA-4. In addition, these results suggest that refolded B7-2 is biologically active and may be a useful therapeutic and experimental reagent for regulating T cell activity. Refolded and purified B7-2 was crystallized by the hanging-drop vapor diffusion method, allowing for the initiation of an X-ray crystallographic study.
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Xuewu
authors.initials: X
authors.lastname: Zhang
authors.forename: Jean-Claude D
authors.initials: JC
authors.lastname: Schwartz
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Stanley G
authors.initials: SG
authors.lastname: Nathenson
mesh_terms: D000069594:Abatacept; D015703:Antigens, CD / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D000943:Antigens, Differentiation / Q000737:chemistry; D051940:B7-2 Antigen; D001665:Binding Sites; D018106:CD28 Antigens / Q000378:metabolism; D060908:CTLA-4 Antigen; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D051940:B7-2 Antigen; D018106:CD28 Antigens; C497964:CD86 protein, human; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D004220:Disulfides; D018796:Immunoconjugates; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D000069594:Abatacept