کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2021297 1069239 2007 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
High-level expression and purification of the human bradykinin B2 receptor in a tetracycline-inducible stable HEK293S cell line
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
High-level expression and purification of the human bradykinin B2 receptor in a tetracycline-inducible stable HEK293S cell line
چکیده انگلیسی

The B2 bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B2 receptor for future biophysical studies. Different tagged B2 receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B2 receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60 pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B2 receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B2 receptor with tetracycline and sodium butyrate led to a level of 100 pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-β-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B2 receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 55, Issue 2, October 2007, Pages 300–311
نویسندگان
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