Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Autor: Elegheert J; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. jonathan.elegheert@u-bordeaux.fr.; Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France. jonathan.elegheert@u-bordeaux.fr.; Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France. jonathan.elegheert@u-bordeaux.fr., Behiels E; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.; Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.; Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France., Bishop B; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., Scott S; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.; MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK., Woolley RE; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., Griffiths SC; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., Byrne EFX; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.; Department of Bioengineering, Stanford University, Stanford, CA, USA., Chang VT; MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK., Stuart DI; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., Jones EY; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., Siebold C; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. christian@strubi.ox.ac.uk., Aricescu AR; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK. radu@mrc-lmb.cam.ac.uk.; MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK. radu@mrc-lmb.cam.ac.uk.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2018 Dec; Vol. 13 (12), pp. 2991-3017.
DOI: 10.1038/s41596-018-0075-9
Abstrakt: Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO 2 , for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABA A R) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.
Databáze: MEDLINE
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