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.
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