HIGH-LEVEL EXPRESSION OF GLUCAGON AND GLUCAGON-LIKE PEPTIDE 1 RECEPTORS IN TETRACYCLINE-INDUCIBLE STABLE HEK293S GNTI-CELLS

摘要

The lack of high-resolution, three-dimensional information is an important factor that limits our understanding of the molecular basis of peptide ligand binding and subsequent development of potential drugs. Despite insights from structure-function studies a detailed 3-d structure of receptor-bound peptide hormone will ultimately come from NMR and X-ray analysis. These studies require relatively large amounts of purified receptor and most GPCRs are notoriously difficult to overexpress and isolate in functional form. We have established and characterized a novel tetracycline-inducible system in stable HEK293S GnTI~- cells for the high-level expression of both human glucagon (hGR) and glucagon-like peptide 1 (hGLP-1R) receptors . Using a strategy frequently used in bacterial expression systems, the membrane proteins are expressed by induction of the desired gene only after the cells have grown to near-maximum density. In addition, these cells lack N-acetylglucosaminyltransferase I activity and do not allow formation of heterogeneous N-glycans that may complicate crystallization protocols and NMR analysis. To facilitate single-step, immunoaffinity purification of hGR and hGLP-1R, the rhodopsin 1D4 peptide tag TETSQVAPA was engineered at the extreme C-terminus of each receptor.