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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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.
The authors declare no competing interests.
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Key references using this protocol
Chang, V. T. et al. Nat. Immunol. 17, 574–582 (2016): https://www.nature.com/articles/ni.3392
Integrated supplementary information
LTR: long-terminal repeat; ψ: psi packaging signal; RRE: Rev response element; cPPT: central polypurine tract; CMV-MIE: major immediate early cytomegalovirus enhancer/promoter; SS: signal sequence; MCS: multiple cloning site; 3C: Human Rhinovirus (HRV) 3C protease cleavage site; Avi: Avi-tag; His6: His6-tag; WPRE: Woodchuck Hepatitis Virus posttranscriptional regulatory element; ΔU3: U3 deletion in the 3ʹ LTR; AmpR: Ampicillin resistance gene; ori: origin of replication; SV40: Simian vacuolating virus 40; UTR: untranslated region. The genetic elements flanked by the 5ʹ and 3ʹ LTRs will be stably integrated into the host cell genome as proviral DNA, and are highlighted in pink. The gene encoding E. coli xanthine-guanine phosphoribosyltransferase (XGPRT, indicated with a star symbol) has been inactivated by deletion of a single base pair, likely to remove an internal KpnI site, and resulting introduction of a stop codon. This modification was already present in the pHR-SIN-CSGW lentiviral transfer plasmid on which the pHR-CMV-TetO2 plasmid suite is based. The dominant selectable marker XGPRT is normally used to permit isolation of stably transfected cells using mycophenolic acid, an immunosuppressant drug. We did not correct the open reading frame since our procedure does not require stable maintenance of the lentiviral transfer plasmid in HEK293T Lenti-X cells. The figure was prepared using the SnapGene software (from GSL Biotech; available at http://www.snapgene.com/).
The pHR-CMV-TetO2 multiple cloning site (MCS) is fully compatible with subcloning of inserts from pHLsec; XbaI/EcoRI – cRPTPσ signal sequence – AgeI – insert – KpnI – tag + stop codons – XhoI, as well as with in vivo assembly (IVA) cloning. (A) The pHR-CMV-TetO2_3C-Avi-His6(_IRES-FP) plasmids contain the 3C-Avi-His6 tag inserted between KpnI and XhoI. (B) The pHR-CMV-TetO2_3C-Twin-Strep(_IRES-FP) plasmids contain the 3C-Twin-Strep tag inserted between KpnI and XhoI. The figure was prepared using the SnapGene software.
Schematic representation of the empty pHR-CMV-TetO2 (4.2 kb proviral DNA), pHR-CMV-TetO2_IRES-FP (5.5 kb proviral DNA), pHR-CMV-TetO2_3C-mVenus-Twin-Strep (5.0 kb proviral DNA), pHR-CMV-TetO2_3C-Avi-His6_HA-BirA-ER (5.9 kb proviral DNA), pHR-SFFV (4.0 kb proviral DNA) and pHR-CAG (5.2 kb proviral DNA) transfer plasmid variants. ψ: psi packaging signal; RRE: Rev response element; PPT: polypurine tract; CMV-MIE: major immediate early cytomegalovirus enhancer/promoter; SFFV: Spleen Focus Forming Virus; CAG: CMV enhancer, chicken β-actin promoter, rabbit β-globin splice acceptor; TRE: tetracycline response element; MCS: multiple cloning site; IRES: internal ribosomal entry site; FP: fluorescent protein; WPRE: Woodchuck Hepatitis Virus posttranscriptional regulatory element; LTR: long-terminal repeat; 3C: Human Rhinovirus (HRV) 3C protease site; mV: mVenus; TS: Twin-Strep. The figure was prepared using the SnapGene software.
(A) Schematic representation of the mRuby2-IRES-EmGFP expression construct (2.1 kb insert, 6.0 kb proviral DNA). (B) Non-concentrated lentiviral particles encoding mRuby2-IRES-EmGFP were used to infect HEK293T cells. IRES-EmGFP mean fluorescence intensity is ~30-fold lower at identical 488 nm laser power as compared to free EmGFP expression. Scale bar: 100 μm. (C) EmGFP and mRuby2 co-localize and their fluorescence intensities show a Pearson correlation coefficient (Pearson’s r) of 0.89 between pixel values, implying that IRES-EmGFP is predictive of the presence and expression level of the preceding transgene. Free and bicistronic mRuby2 mean fluorescence intensities are comparable, indicating that IRES-EmGFP does not negatively affect expression of the preceding transgene. Fluorescence images were taken on a Leica SP8 SMD X confocal microscope as sequential scans to avoid spectral crosstalk of EmGFP and mRuby fluorescence into the red and green detection channels, respectively. Fluorescence emission was detected using hybrid detectors operated in photon-counting mode, meaning that every incident photon gave rise to one gray value. Scale bar: 100 μm. (D) Non-concentrated lentiviral particles encoding mRuby2-IRES-EmGFP were used to infect HEK293S GnTI– TetR cells. Expression was induced using 1 μg/mL Dox for 12 h. Using flow cytometry, we measured a Pearson’s r of 0.85 between EmGFP and mRuby2 compensated fluorescence intensities, based on a total of 35,546 single, gated cells.
The assay was performed as described in Box 2 of the main Protocol. Functional lentiviral titer (and thus MOI) decreases with increasing insert size. Images were taken on a Nikon wide-field TE2000U Microscope. Exposure time was 500 ms and every image was individually enhanced using ImageJ to reveal all cells with fluorescence. Scale bar: 200 μm.
Non-concentrated lentiviral particles encoding transgene-IRES-EmGFP were used to infect HEK293T cells. The plots show the forward and side scatter area (A), height (H) and width (W), and fluorescence gates used to analyse cells expressing EmGFP. Functional lentiviral titer (and thus MOI) decreases with increasing insert size, leading to decreasing transduction efficiency and also a lower number of integrated gene copies per cell.
(A) The mVenus-GABAAR-β3FL-1D4 construct was cloned into the pHR-CMV-TetO2 transfer plasmid. The FSEC analysis shows lentivirus-mediated protein expression in function of expression time (24, 48 h or 72 h), expression temperature (30°C or 37°C), concentration of Dox (0, 5 or 10 μg/mL), and concentration of VPA or sodium butyrate (0 or 5 mM). (B) The mVenus-GABAAR-β3FL-1D4 construct was cloned into a modified pEZT-BM plasmid. The FSEC analysis shows BacMam-mediated protein expression in function of expression time (24, 48 h or 72 h), expression temperature (30°C or 37°C), concentration of VPA or sodium butyrate (0 or 5 mM), and multiplicity of infection (MOI; 0, 1, 4 or 10). Note the difference in scale for the fluorescence emission between panels A and B.
(A) Non-concentrated lentiviral particles encoding either mTurquoise2 (mT2; 0.7 kb insert, 4.7 kb proviral DNA), mVenus (mV; 0.7 kb insert, 4.7 kb proviral DNA) or mRuby2 (mR2; 0.7 kb insert, 4.7 kb proviral DNA) were used to co-infect HEK293S GnTI– TetR cells. Fluorescence microscopic imaging of separately infected or co-infected cells, before and after induction with 10 μg/mL Dox, and after 24 h of expression. DIC; differential interference contrast. Fluorescence images were taken on a Leica SP8 SMD X confocal microscope as sequential scans to avoid spectral crosstalk of fluorophores into the separate detection channels. (B) The plots show the forward and side scatter area (A), height (H) and width (W), and fluorescence gates used to isolate cells expressing mTurquoise2, mVenus and mRuby2. Scale bar: 200 μm.
About this article
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