Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins
Nat Protoc. 2018-11-19; 13(12): 2991-3017
DOI: 10.1038/s41596-018-0075-9
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1. Nat Protoc. 2018 Dec;13(12):2991-3017. doi: 10.1038/s41596-018-0075-9.
Lentiviral transduction of mammalian cells for fast, scalable and high-level
production of soluble and membrane proteins.
Elegheert J(1)(2)(3), Behiels E(4)(5)(6), Bishop B(4), Scott S(4)(7), Woolley
RE(4), Griffiths SC(4), Byrne EFX(4)(8), Chang VT(7), Stuart DI(4), Jones EY(4),
Siebold C(9), Aricescu AR(10)(11).
Author information:
(1)Division of Structural Biology, Wellcome Centre for Human Genetics, University
of Oxford, Oxford, UK. .
(2)Interdisciplinary Institute for Neuroscience, University of Bordeaux,
Bordeaux, France. .
(3)Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France.
.
(4)Division of Structural Biology, Wellcome Centre for Human Genetics, University
of Oxford, Oxford, UK.
(5)Interdisciplinary Institute for Neuroscience, University of Bordeaux,
Bordeaux, France.
(6)Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France.
(7)MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge,
UK.
(8)Department of Bioengineering, Stanford University, Stanford, CA, USA.
(9)Division of Structural Biology, Wellcome Centre for Human Genetics, University
of Oxford, Oxford, UK. .
(10)Division of Structural Biology, Wellcome Centre for Human Genetics,
University of Oxford, Oxford, UK. .
(11)MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge,
UK. .
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.
DOI: 10.1038/s41596-018-0075-9
PMID: 30455477