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Expression, Refolding, Purification, Molecular Characterization, Crystallization, and Preliminary X-ray Analysis of the Receptor Binding Domain of Human B7-2
- Source :
-
Protein Expression & Purification . Jun2002, Vol. 25 Issue 1, p105. 9p. - Publication Year :
- 2002
-
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. [Copyright &y& Elsevier]
- Subjects :
- *IMMUNE response
*T cells
*CRYSTALLIZATION
Subjects
Details
- Language :
- English
- ISSN :
- 10465928
- Volume :
- 25
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Protein Expression & Purification
- Publication Type :
- Academic Journal
- Accession number :
- 8505854
- Full Text :
- https://doi.org/10.1006/prep.2002.1616