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

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-TetO2, 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 (GABAAR) 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.

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The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank G. Davies and C. Green (University of Oxford) for assistance with flow cytometry, S. Padilla-Parra and L.A.J. Alvarez (University of Oxford) for assistance with microscopic imaging, D. Laverty (MRC-LMB) for the modified pEZT-BM vector, J. Watson (MRC-LMB) for advice on IVA cloning, and S. Ressl (Indiana University Bloomington) for comments on the manuscript. pMD2.G (Addgene plasmid no.12259) and psPAX2 (Addgene plasmid no. 12260) were a gift from D. Trono (École Polytechnique Fédérale de Lausanne (EPFL)). This work was supported by a Marie-Curie (FP7-328531) long-term postdoctoral fellowship (to J.E.); UK Medical Research Council grants MR/L009609/1 and MC_UP_1201/15 (to A.R.A.) and MR/L017776/1 (to C.S.); a UK Biotechnology and Biological Sciences Research Council grant (BB/M024709/1, to A.R.A.); Cancer Research UK grants C20724/A14414 (to C.S.) and C375/A10976 (to E.Y.J.); a European Research Council (ERC) grant (647278, to C.S.); and Wellcome Trust studentships (105247/Z/14/Z, to S.S., and 203726/Z/16/Z, to R.E.W.). The Wellcome Centre for Human Genetics is funded by Wellcome Trust Core Award 203852/Z/16/2.

Author information

  1. Jonathan Elegheert & Ester Behiels Present address: Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France
  2. Eamon F. X. Byrne Present address: Department of Bioengineering, Stanford University, Stanford, CA, USA
  3. These authors contributed equally: Ester Behiels, Benjamin Bishop.

Authors and Affiliations

  1. Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Jonathan Elegheert, Ester Behiels, Benjamin Bishop, Suzanne Scott, Rachel E. Woolley, Samuel C. Griffiths, Eamon F. X. Byrne, David I. Stuart, E. Yvonne Jones, Christian Siebold & A. Radu Aricescu
  2. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK Suzanne Scott, Veronica T. Chang & A. Radu Aricescu
  3. Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France Jonathan Elegheert & Ester Behiels
  1. Jonathan Elegheert