Preparation and crystallization of the disulfide-linked HLA-G dimer |
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Authors: | Shiroishi Mitsunori Kohda Daisuke Maenaka Katsumi |
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Institution: | Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan. |
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Abstract: | HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dimer both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-A data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines. |
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