title: Generation of Tetracycline-Inducible Mammalian Cell Lines by Flow Cytometry for Improved Overproduction of Membrane Proteins.
abstract: Overexpression of mammalian membrane proteins in mammalian cells is an effective strategy to produce sufficient protein for biophysical analyses and structural studies, because the cells generally express proteins in a correctly folded state. However, obtaining high levels of expression suitable for protein purification on a milligram scale can be challenging. As membrane protein overexpression often has a negative impact on cell viability, it is usual to make stable cell lines where the protein of interest is expressed from an inducible promoter. Here we describe a methodology for optimizing the inducible production of any membrane protein fused to GFP through the isolation of clonal cell lines. Flow cytometry is used to sort uninduced cells and the most fluorescent 5 % of the cell population are used to make clonal cell lines.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Juni
authors.initials: J
authors.lastname: Andréll
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Edwards
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Daly
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK. cgt@mrc-lmb.cam.ac.uk.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Tate
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002999:Clone Cells / Q000166:cytology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; D013752:Tetracycline
keywords: Flow cytometry; GFP; GPCR; HEK293 cell line; Membrane protein; Overexpression; Transporter