Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

Abstract

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol we show how to use small-scale transient transfection and fluorescence-detection, size-exclusion chromatography (FSEC) experiments using a GFP-His₈ tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI⁻ (N-acetylglucosaminyltransferase I-negative) cells in suspension culture, and over-express the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl), for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

INTRODUCTION

Since the initial observation that insertion of a human cytomegalovirus (CMV) promoter or a Rous sarcoma virus (RSV) promoter into an Autographa californica multiple nucleopolyhedrosis virus (AcMNPV; from here on referred to as baculovirus) transfer vector allowed for expression of foreign genes in hepatocytes and other mammalian cell lines ^1,2, baculovirus -mediated gene transfer into mammalian cells (BacMam) has been employed for a growing number of applications. These applications include drug discovery (identification and development of new therapeutic agents) through recombinant protein expression for cell-based functional assays using G-protein-coupled receptors^3,4, nuclear receptors⁵, ion channels^6,7 and ATP-binding cassette drug transporters⁸. More recently, BacMam has been used for large-scale protein production for crystallography^9–20. The success of these applications, however, depends in part on the efficient production and amplification of baculovirus and on subsequent large-scale transduction and heterologous protein expression. In addition to these challenges, obtaining sufficient quantities of membrane protein for crystallography is frequently compounded by low levels of expression and instability of the candidate membrane protein, thus requiring screening of many constructs. Furthermore, some mammalian membrane proteins require specific post translational modifications and a near native lipid environment, thus rendering expression in insect cells or in yeast untenable. Taken together, these complexities can result in a high cost for heterologous membrane protein expression in mammalian cells and thus improving the efficiency of the process is important.

Here we describe methods to screen constructs and to optimize heterologous expression of membrane proteins from BacMam transduced HEK293S GnTI⁻ (N-acetylglucosaminyltransferase I-negative) cells for purification and crystallization (Fig. 1). We have constructed a vector (pEG BacMam) for high level expression in mammalian cells using elements derived from a previously described vector, pVLAD¹⁰. Once genes of candidate membrane proteins are fused in frame with a GFP tag and cloned into pEG BacMam, they can be rapidly screened for expression and monodispersity using transient transfection in adherent cells coupled with fluorescence-detection size-exclusion chromatography (FSEC)^21,22. We also optimized virus amplification and protein expression protocols such that cost and time for expressing most membrane proteins in HEK293 GnTI⁻ cells is similar to or better than expression in Sf9 cells.

Figure 1.

Flow chart of the overexpression of membrane proteins in HEK293S GnTI⁻ cells. After one or more rounds of screening, a few potential candidates are chosen for large-scale protein expression.

In the current protocol we provide two examples in which we express Gallus gallus Acid-sensing ion channel 1a^16,23(cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in mammalian cells^24,25. After optimizing protein expression, we compared the expression of cASIC1 and GluCl in mammalian cells and insect cells. We show that five-fold more GluCl pentamer can be obtained in mammalian cells. In the case of cASIC1, not only can two-fold more trimer be obtained in mammalian cells, but also the protein is more monodisperse and experiences less spontaneous cleavage of the GFP-His₈ tag. This protocol is now in standard use in our laboratory for mammalian-expressed membrane proteins^15–17,20.

Development of the protocol

To increase the heterologous expression of challenging membrane proteins we first constructed pEG BacMam for high-level protein expression in mammalian cells with the ability to express multiprotein complexes from a single vector (Fig. 2). To do this we chemically synthesized genetic elements derived from the previously described BacMam vector, pVLAD¹⁰, which include a strong CMV promoter for robust transcription, a synthetic intron for efficient RNA splicing and mRNA processing and a WPRE motif for efficient mRNA processing, stability and export. These chemically synthesized elements were combined with the pFBDM²⁶, a bicistronic vector with a restriction enzyme module that allows the assembly of multiple expression cassettes, to generate pEG BacMam. After the gene of interest is cloned into pEG BacMam, we screen constructs by small scale transfection/FSEC before moving to the time consuming process of virus amplification²¹.

Figure 2.

Map of BacMam Expression Vector. For expression in mammalian cells, genes of interest are cloned into the multiple cloning site behind the CMV promoter using unique restriction sites. Elements that are important for high level expression are shown, including those that are important for transcription initiation (CMV promoter), transcription termination (SV40 poly A late signal), mRNA processing (intron and WPRE motif). Also indicated are the elements important for insect cell expression and baculovirus amplification including promoters (polh and p10), terminators (SV40 and HSVtk), transposon elements (Tn7L and Tn7R), and resistance markers (ampicillin and gentamicin).

Although other HEK cell lines can be used, for screening and expression we typically use HEK293S GnTI⁻ cells, a mammalian cell line that expresses proteins that are more mannose-rich and as a result are easily removed with endoglycosidases such as EndoH²⁷. Although the use of these cells and EndoH can reduce heterogeneity caused by complex glycans that can create problems in crystallographic studies, it may not beneficial for every protein. Therefore, it is advantageous to test protein expression in other mammalian cell lines as well as determine if the use of EndoH affects the solubility and the heterogeneity of the glycoprotein.

To determine the expression level and monodispersity of the candidate membrane protein, HEK293S GnTI⁻ cells are transfected with the pEG BacMam plasmid containing the gene of interest; harvested after 48 hours; and then solubilized in a buffer containing n-dodecyl-β-D-maltoside (C₁₂M), maltose-neopentyl glycol²⁸ (MNG-3), or other detergent. The resulting supernatant is analyzed by FSEC (Fig. 3). As shown in Figure 3, removing the 64 residues from the carboxy terminus of cASIC1 (cASIC463) increases monodispersity and reduces cleavage of the GFP-His₈ tag. In addition to removing flexible termini, there are many methods that can be used to optimize expression and stability of proteins including codon optimization, surface entropy reduction, and thermostability mutations^29–31. Small scale transfection followed by whole cell solubilization and FSEC allows the screening of hundreds of candidates in ≤1 month.

Figure 3.

Screening constructs by small scale transient transfection in HEK293S GnTI⁻ cells and FSEC. 1 μg of N-terminal eGFP tagged full length cASIC1 or cASIC463 (cASIC1 truncated 64 residues from the carboxy termini) subcloned into pEG BacMam was transfected separately into HEK293S GnTI⁻ cells, as described in the text. Cells were harvested 48 h after transfection and solubilized extracts were analyzed by FSEC to determine the behavior of the fusion proteins. c.p.s., counts per second.

Once a promising candidate is identified, the plasmid is transformed into the DH10Bac E. coli strain to generate the recombinant bacmid DNA which is then used to transfect insect cells to generate BacMam virus. We have detailed our methods for isolation of bacmid DNA, transfection of Sf9 cells and baculovirus amplification that we use to reduce costs and ensure good quality BacMam virus. We have found for some constructs that the multiplicity of infection (MOI) during virus amplification is 10 to 100 fold below the range recommended by the Bac-to-Bac system protocol (http://www.lifetechnologies.com/us/en/home/life-science/protein-expression-and-analysis/protein-expression/insect-expression/bac-to-bac-baculovirus-expression-system.html). We have also found that a low MOI (MOI of 2 or less) is sufficient for mammalian cell transduction and that too much virus results in low cell numbers, possibly due to too much Sf9 medium or virus added to the culture. Therefore, before virus amplification or transduction of mammalian cells for protein expression, virus titer should be determined using the end point dilution assay³², flow cytometric assay^33,34, or the viral plaque assay³⁵.

In addition to MOI, we also explored different growth and expression conditions for BacMam transduced HEK293S GnTI⁻ cells to boost protein expression. After testing several types of media for growth of suspension cells, we found that the use of Gibco FreeStyle 293 Expression Medium (Invitrogen) allowed for increased growth rates and reduced cell clumping of HEK293S GnTI⁻ cells in suspension. To further minimize cell clumping, we also assessed the growth of suspension cells in different vessels, including square bottles, flat-bottom flasks and baffled Erlenmeyer flasks. We found that baffled flasks minimized cell clumping and promoted cell growth. To reduce costs, the polycarbonate Erlenmeyer flasks can be washed, autoclaved and used again up to 20 times. If after autoclaving the filter in the cap deteriorates, a replacement cap can be purchased.

Previously it has been shown that lowering the temperature post-transduction or transfection enhances protein expression in mammalian cells^36–43. In a time course experiment for cASIC1, we found that at the optimal harvest time of 72h post transduction there was a 4.3 fold increase in expression at 30 °C compared to 37 °C (Fig. 4a). Gaussian fitting, as described previously^21,22, was performed on the FSEC profiles to determine the peak area of cASIC1 trimer. A graph of the trimer peak area shows that the expression of cASIC1 is higher at 30 °C than at 37 °C for most time points (Fig. 4b). To monitor the expression of GluCl in HEK293S GnTI⁻ cells, an EGFP GluCl gene fusion^25,44 was cloned into pEG BacMam and used to generate BacMam virus. At the optimal harvest time of 72 h post transduction for cells expressing GluCl, there was a 9.5-fold increase in expression at 30 °C with higher expression of GluCl at all time points at 30 °C (Fig. 4c and 4d). To show that the protein present in the major peaks for cASIC and GluCl are the expected molecular weight, peak fractions were collected and analyzed by SDS-PAGE followed by Western blot analysis using an antibody against GFP (Fig. 4e). In fact, for the expression of most proteins in HEK293S GnTI⁻ cells, we have found that lowering the temperature during expression increases protein yields at least two-fold. In some cases, lowering the temperature is essential to obtain monodisperse, well-folded protein.

Figure 4.

Time course of expression of cASIC and GluCl in HEK293S GnTI⁻ cells. HEK293S GnTI⁻ cells (3×10⁶ cells/ml) were transduced with BacMam virus for cASIC463 (a and b) or GluCl-EGFP (c and d) at a MOI of 1 and incubated at 37 °C. After 8 h, 10 mM sodium butyrate was added and cultures were left at 37 °C or shifted to 30 °C. Time points were taken at the indicated times, samples were frozen and later solubilized with 40 mM C₁₂M in TBS pH 8 and analyzed by FSEC. (a and c) Representative FSEC profiles from cASIC463 (a) and GluCl-EGFP (c) detected by GFP fluorescence. (b and d) Comparison of expression at 37 °C and 30 °C from cASIC463 and (b) GluCl-EGFP (d) based on the peak area estimated by Gaussian fitting. (e) Western blot analysis using an anti-GFP polyclonal antibody of peak fractions isolated from FSEC profiles from (a) cASIC463 and (c) GluCl-EGFP.

Finally, the use of histone deacetylase inhibitors has been shown to enhance protein expression in HEK293S cells^10,45. We found that for most membrane proteins histone deacetylase inhibitors boost expression. For cASIC1 there is a 7-fold increase in expression at 72 h post-transduction as well as higher expression of cASIC1 at all time points when HEK293S GnTI⁻ cells are treated with 10 mM sodium butyrate (Fig. 5a and 5b). Previously published data suggests that valproic acid is more efficient than sodium butyrate at enhancing recombinant protein production from mammalian cells⁴⁵. However, we find that both sodium butyrate and valproic acid enhance protein expression from BacMam transduced HEK293S GnTI⁻ cells (Fig. 5c). Typically sodium butyrate is added to cultures between 8–24 hours post-transduction; however the amount and time of sodium butyrate addition should be optimized for each protein. Overall optimization of conditions (construct, MOI, cell density, temperature and harvest time of BacMam transduced HEK293S GnTI⁻ cells) could either increase expression or decrease aggregation leading to more properly folded protein and therefore the most favorable conditions for each protein should be determined before attempting a large scale expression.

Figure 5.

Effect of histone deacetylase inhibitors on the expression of cASIC and GluCl in HEK293S GnTI⁻ cells. (a and b) Comparison of cASIC expression with and without sodium butyrate. Representative FSEC profiles from cASIC463 detected by GFP fluorescence (a) and comparison of expression from cASIC463 based on the peak area estimated by Gaussian fitting (b) in the presence or absence of sodium butyrate. (c) Representative FSEC profiles from GluCl-EGFP in the presence of sodium butyrate or valproic acid.

Other applications of the method

The protocol described here could also be used to optimize the expression of heteromers or protein complexes in mammalian cells. One option to simultaneously express multiple proteins is to co-infect with multiple BacMam viruses (with an optimized MOI for each virus). Alternatively, pEG BacMam could be used to express multiprotein complexes by combining two pEG BacMam plasmids using unique restriction enzyme sites. As a result, multiple genes could be transduced by a single BacMam virus allowing for the simultaneous expression of two or more genes.

Some variants of pEG BacMam also contains vesicular stomatitis Indiana virus glycoprotein (VSIV-G) under control of the P10 promoter (a baculovirus specific promoter). VSIV-G is a viral capsule protein important for mediating viral entry and has been shown to increase the transduction efficiency of baculovirus for some mammalian cells⁴⁶. The P10 promoter could be used to drive the expression of VSIV-G in insect cells allowing incorporation of VSIV-G into the baculovirus to enhance transduction from other mammalian cell lines, such as the human lung carcinoma line A549 and the human hepatoma lines HepG2 and HuH7⁴⁶.

Comparison to other methods

Many methods have been utilized for overexpression of mammalian proteins including plasmid transfection, stable cell lines and viral expression systems such as Sindbis virus, vaccinia virus, and Semliki Forest virus, and AcMNPV⁴⁷. Compared to BacMam, each of these methods has advantages and disadvantages in terms of cost, time, efficiency, safety and reproducibility.

One such transient expression method involves transfection of plasmid DNA into adherent cells or cultures and overexpression is either immediate or induced, depending on the promoter. Plasmid transfection is fast, safe and easy to use for high throughput screening^48–50. If commercial transfection reagents and plasmid isolation kits are used, however, plasmid transfection can be expensive for large scale expression. Furthermore, the level of expression using plasmid transfection can be limited by the plasmid size, the number of plasmids transfected, and cytotoxic effects that have been observed with many transfection reagents. The use of BacMam can be cheaper than plasmid transfection for large scale expression and multiple rounds of expression. In addition, BacMam is not limited by the gene number or size. In our hands the level of protein expression, especially multi-subunit proteins, is higher using BacMam than plasmid transfection.

Stable expression in mammalian cells requires the integration of a transfected transgene into the cell’s genome using Geneticin or other selection methods. Once clonal cell lines are generated and sorted for high-level producers, long-term overexpression from stably transfected cells can be robust, easy and consistent^51,52. Furthermore, stable cell lines can be generated using regulated expression, such as the tetracycline-inducible expression system, thus allowing for large scale expression of proteins cytotoxic to the cell^51,53. Although stable expression enables the production of large quantities of protein, when compared to BacMam generating a stable cell line is time consuming.

Although other viral expression systems for protein expression such as lentiviruses⁵⁴, adenoviruses⁵⁵, Sindbis virus^56,57, vaccinia virus⁵⁸, and Semliki Forest virus^59,60 (SFV) exist, SFV has been used to express a large number of membrane proteins consisting of mostly GPCRs⁴⁷. To make virus, candidate genes are cloned into SFV plasmid and used as template for RNA synthesis. The RNA is then co-transfected (either using electroporation or liposome reagents) with helper RNA and packaged into SFV particles that can then be used to infect cells in culture. While SFV can be easily used for small scale studies by transfecting synthesized RNA into cells and analyzing expression, using SFV for large cultures is more challenging than BacMam due to the amount of RNA that is needed to make virus for large scale studies.

An effective method using BacMam was recently described to produce milligram quantities of proteins sufficient for crystallization¹⁰. However, the methods outlined therein to produce recombinant BacMam virus using the BD BaculoGold system are not as cost efficient as the Bac-to-Bac method if multiple constructs are expected to be generated. In addition, we optimized growth and expression conditions using BacMam in order to express sufficient amounts of our desired membrane proteins.

Another commonly used method for overexpression of proteins is AcMNPV baculovirus infection of insect cells. Although insect cells such as Sf9 cells have been used in our lab previously to provide sufficient protein for crystallization studies^25,61–64, some membrane proteins require a near-native environment to help ensure functional expression. The advantages for expression of eukaryotic membrane proteins in mammalian cells over Sf9 include improved post-translational modifications such as N-linked glycosylation^65,66 and a different lipid environment that contains higher amounts of cholesterol. We performed a side-by-side comparison of protein expression from insect cells and mammalian cells. We found a two-fold increase in cASIC1 trimer and an increase in homogeneity (Fig. 6a-b) and that five-fold more GluCl pentamer can be obtained in mammalian cells (Fig. 6c-d).

Figure 6.

Comparison of expression time course in HEK293S GnTI⁻ cells and Sf9 cells. HEK293S GnTI⁻ cells were transduced with BacMam virus for cASIC463 (a and b) or GluCl-EGFP (c and d) as described above. Sf9 cells (3×10⁶ cells/ml) were infected with baculovirus cASIC463 (a and b) or GluCl-EGFP (c and d) and placed at 27 °C. After 18 h, Sf9 cultures were left at 27 °C or shifted to 20 °C. Time points were taken, frozen and solubilized as described above. (a and c) Representative FSEC profiles from cASIC463 (a) and GluCl-EGFP (c) detected by GFP fluorescence. (b and d) Comparison of expression from cASIC463 (b) GluCl-EGFP (d) based on the peak area estimated by Gaussian fitting.

MATERIALS

REAGENTS

• pEG BacMam plasmid containing the gene of interest in frame with a GFP tag and a tag appropriate for affinity chromatography (such as a His₈ tag) cloned downstream of the CMV promoter (Fig. 2)

• HEK293S GnTI⁻ cells (ATCC, cat. no. CRL-3022)

• Gibco Freestyle 293 Expression medium (Life Technologies, cat. no. 12338-018)

• US Certified Gamma Irradiated FBS (Life Technologies, cat. no. 0984018DJ)

• DMEM (with 4.5 g/l glucose, L-glutamine, and sodium pyruvate; Corning|Cellgro, cat. no. 10-013)

• Opti-MEM I Reduced Serum Medium (Life Technologies, cat. no. 31985-088)

• Lipofectamine 2000 (Life Technologies, cat. no. 11668-027)

• PBS (without calcium and magnesium; Corning|Cellgro, cat. no. 21-040-CM)

• Trypsin/EDTA (Corning|Cellgro, cat. no. 25-052-CV)

• Tris base (Fisher, cat. no. BP152)

• NaCl (Sigma, cat. no. 59888)

• C₁₂M (Affymetrix, cat. no. D310)

• PMSF (Sigma, cat. no. 78830)

• Leupeptin (Sigma, cat. no. L0649)

• Aprotinin (Sigma, cat. no. A1153)

• Pepstatin (Sigma, cat. no. P4265)

• Sodium butyrate (Sigma, cat. no. 303410)

• Valproic Acid (Sigma, cat. no. P4543)

• DH10Bac competent cells (Life Technologies, cat no. 10361-012)

• Sf9 Easy Titer cell line³²

• Cellfectin II reagent (Life Technologies, cat. no. 10362-100)

• Sf9 cells (Life Technologies, cat. no. 12659017)

• Sf-900 III SFM media (Life Technologies, cat. no. 12658-027)

• QIAprep Spin Miniprep Kit (QIAGEN, cat. no. 27104)

• HyClone SFX-Insect Media (GE/Hyclone, cat. no. SH30278.02)

• G 418 disulfate salt (Sigma, cat. no. A1720-1g)

• Trypan blue solution (Corning|Cellgro, cat. no. 25-900-CL)

EQUIPMENT

• Incubator (Thermo Scientific, cat. no. 3950)

• Tissue culture plate (100 mm; BD Falcon, cat. no. 35300)

• Tissue culture plate (6 well; BD Falcon, cat. no. 353046)

• Erlenmeyer baffled Flasks (2000 ml; BioExpress, cat. no. F-5909-2000B)

• Nunc EasYFlask (75 cm², Filter Cap; Thermo Scientific, cat. no. 156499)

• Syringe filters (PES 13 mm Diameter, 0.22 um, PP housing; Argos, cat. no. FE12S)

• Filter systems (250 ml, 0.22 μm; Corning, cat. no. 430767)

• Virus counter 2100 (ViroCyt)

• Optima TL Ultracentrifuge (Beckman)

• Fluorescence-detection size-exclusion chromatography (FSEC)²¹

• Tissue culture plate (Costar 96-Well Black Clear-Bottom Plates; Costar /Corning, cat. no. 3603)

• Sterile Disposable Reagent Reservoirs (50ml white; Costar /Corning, cat. no. 4870)

• Cluster Tube System 8-tube strip (Costar /Corning product, cat. no. 4413)

• Cell Scrapers (Handle 18cm blade 1.8cm; Corning, cat. no. 353085)

• Hemocytometer (Hausser Scientific, cat. no. 1492)

REAGENT SETUP

DMEM medium

To 500 ml of DMEM medium, add 50 ml of FBS. Store at 4 °C for a month.

Suspension medium

To 1 liter of Freestyle 293 Expression medium, add 20 ml of FBS. Store at 4 °C for a month.

Sf9 easy medium

To 1 liter of HyClone SFX-Insect Media, add 50 ml of FBS and 150 μg/ml G418. Store at 4 °C for a month.

Sodium butyrate (2M)

Dissolve 11 g of sodium butyrate with water to a final volume of 50 ml and filter-sterilize using a 0.2 μm filter inside the biological safety cabinet. Store at −20 °C for at least 1 month.

Tris-buffered saline (TBS)

Mix 20 mM Tris HCl (pH 8), 150 mM NaCl. Store at RT; 25 °C for at least a month.

Solubilization buffer

Mix 20 mM Tris HCl (pH8), 150 mM NaCl, and 40 mM C₁₂M. Chill the buffer to 4 °C. Immediately before use add 1 mM PMSF, 200 μM Aprotinin, 2 μg/ml Leupeptin, and 2 μM Pepstatin A. Discard any unused buffer.

FSEC buffer

Mix 20 mM Tris HCl (pH 8), 150 mM NaCl, and 1 mM C₁₂M. Filter using a 0.2 μm filter. Store at 4 °C for up to 1 week.

Purification of plasmid DNA

Purify plasmid DNA using the QIAprep Spin Miniprep Kit (QIAGEN) or another suitable method.

Growth and maintenance of adherent HEK293S GnTI⁻ cells

Cells are cultured as previously described⁵¹.

Growth and maintenance of suspension HEK293S GnTI⁻ cells

HEK293S GnTI⁻ cells are maintained as described in Box 1.

Box 1. Growth and maintenance of suspension HEK293S GnTI⁻ cells ● TIMING 15 min.

• 1|Slough off HEK293S GnTI⁻ cells from a T-75 flask at 80% confluency using 25 ml of FreeStyle293 Expression Medium (Invitrogen) supplemented with 2% FBS.
• 2|Transfer the cell suspension to a 125 ml baffled flask and place it on an orbital shaker within a 37 °C incubator in the presence of 8% CO₂.

  CRITICAL The cell suspension should not exceed more than 40% of the baffled flask size.

• 3|After 24 h dilute the cells to 0.5×10⁶ cells/ml and maintain the suspension adapted cells between 0.2–3 × 10⁶ cells/ml at 40% of vessel size for baffled flasks.

  ? TROUBLESHOOTING

Growth and maintenance of Sf9 cells

Sf9 cells are maintained as suspension cultures at 27 °C in Sf-900 III SFM medium. Isolation of bacmid DNA, transfection of Sf9 cells and amplification of virus are modified methods from the Bac-to-Bac system (Invitrogen) that we use to reduce costs and ensure production of good quality BacMam virus.

Growth and maintenance of Sf9 Easy Titer cell line

Sf9 Easy Titer cell line are maintained as adherent cells at 27 °C in Hyclone SFX-Insect media as described in Box 2.

Box 2. End point dilution assay ● TIMING 1 hr.

The following protocol is for one 96-well black plate that can be used to titer 4 viruses and is based ref. 32. Adjust volumes as necessary.

Growth and maintenance of adherent Sf9 Easy Titer cell line

• 1|When Sf9 Easy Titer cell line reach 90% confluency, aspirate off the medium and wash cells with 5 ml PBS.
• 2|Aspirate off the PBS and add 2 ml of trypsin for 30 sec.
• 3|Add 10 ml of Hyclone SFX-Insect media supplemented with 5 % FBS and 150 μg/ml G418.
• 4|Scrape the cells gently with a sterile cell scraper.
• 5|Dilute cells 1:4 in a 100mm tissue culture dish and incubate at 27 °C.

End point dilution assay

• 6|Using a 100 mm tissue culture dish of Sf9 Easy Titer cells that is 90% confluent, follow Steps 1–4 above.
• 7|Make a 10 ml stock of Sf9 Easy Titer cells at a density of 0.75 × 10⁶ cells/ml in a sterile culture reservoir.
• 8|Using the multi-channel pipet, seed 100 μl/well of 96-well black plate (75,000 cells/well) and let attach for ~15 min at 25 °C.
• 9|Using the sterile, deep-well 8-strip clusters, make 10⁻¹ to 10⁻⁸ stock of virus in Hyclone SFX-Insect media supplemented with 5 % FBS and 150 μg/ml G418 (360 μl medium + 40 μl virus).

  1. 360 μl medium + 40 μl P1 virus = 10⁻¹

  2. 360 μl medium + 40 μl 10⁻¹ dilution = 10⁻²

  3. 360 μl medium + 40 μl 10⁻² dilution = 10⁻³

  4. 360 μl medium + 40 μl 10⁻³ dilution = 10⁻⁴

  5. 360 μl medium + 40 μl 10⁻⁴ dilution = 10⁻⁵

  6. 360 μl medium + 40 μl 10⁻⁵ dilution = 10⁻⁶

  7. 360 μl medium + 40 μl 10⁻⁶ dilution = 10⁻⁷

  8. 360 μl medium + 40 μl 10⁻⁷ dilution = 10⁻⁸

• 10|Once cells have attached to the 96-well plate (~ 20 min incubation at 27 °C), remove medium and replace with the 100 μl diluted virus using a multi-channel pipet.
• 11|Infect each virus in triplicate.
• 12|Incubate the 96-well plate at 27 °C.
• 12|
  At 72 hr post-infection, count the number of green foci in the dilution that gives <10 foci/well. To calculate viral titer, use the following equation:

  $$(\text{Average}\text{number}\text{of}\text{foci})\times \text{dilution}\text{factor}\times 10=\text{plaque}\text{forming}\text{units}(\text{pfu})/\text{ml}$$

  ? TROUBLESHOOTING

BacMam virus titer determination

Determine the titer of the BacMam virus using one of the several methods for virus titer determination. We prefer the Sf9 Easy Titer cell line and the endpoint dilution assay³² (see Box 2) or using the flow cytometric assay^33,34.

FSEC

In our laboratory, this is performed as described by Kawate and Gouaux²¹. The analyte is loaded onto a Superose 6 column (10/30, Amersham Biosciences) that has been pre-equilibrated with FSEC buffer. Separation is performed at a flow rate of 0.5 ml/min. The eluent from the SEC column is passed through a Shimadzu fluorometer (RF-20A) fluorometer fitted with a flow cell as described by manufactures instructions. The fluorometer settings are as follows: band-pass, 3 nm/3 nm; excitation, 488 nm; emission, 507 nm; time increment, 1 s; integration time, 1 s; and recording time, 3,000–3,600 s. Calibration with known quantities of GFP have demonstrated that 1–10 ng of GFP can readily be detected.

PROCEDURE

Cell seeding (day 1) ● TIMING 15 min

• 1|Add 1×10⁶ HEK293S GnTI⁻ cells in 2 ml of DMEM, supplemented with FBS to each well of each six-well culture plate. Incubate at 37 °C with 5% CO₂ for 16–24 h.

  ! CAUTION Cell cultures are a potential biohazard. Work in an approved laminar flow hood using aseptic techniques and check institutional and governmental guidelines for recommended protective clothing and proper disposal of waste prior to performing experiments.

  ? TROUBLESHOOTING

Small scale transient transfection to screen constructs (day 2) ● TIMING 45 min

• 2|for each well, prepare an autoclaved 1.5 ml centrifuge tube. Using a pipette, add 4 μl of Lipofectamine 2000 into 50 μl of Opti-MEM I.
• 3|Add 1 μg of Qiagen MiniPrep-purified DNA into 50 μl of Opti-MEM I in a separate 1.5 ml centrifuge tube.
• 4|Add DNA/Opti-MEM I mixture to the Opti-MEM/Lipofectamine mixture, gently mix and incubate for 20 min at RT.
• 5|Pipette the Opti-MEM I-DNA mixture drop wise onto 70–80% confluent HEK293S GnTI⁻ cells. Ensure even dispersal.
• 6|After 8–24 hours, replace the medium with DMEM plus 10 mM sodium butyrate.
• 7|Incubate the cells at 37 °C with 5% CO₂ for 2 days.

Screen constructs by FSEC for monodispersity and expression level (day 4) ● TIMING 3 h

• 8|Aspirate off the medium and wash the transfected adherent cells carefully with 2 ml TBS.
• 9|Add 1 ml TBS to each well, collect the cells and transfer them to a 1.5 ml centrifuge tube.
• 10|Centrifuge the cells at 1,500xg for 5 min, 4 °C.
• 11|Remove the supernatant and resuspend the cell pellet in 200 μl solubilization buffer.
• 12|Nutate samples for 1 h at 4 °C.
• 13|Centrifuge the solubilized sample at 70,000xg in a TL100 ultracentrifuge for 40 min, 4 °C.
• 14|Collect supernatant and analyze 50 μl by FSEC²¹. Allow 1hr for each sample to be analyzed by FSEC. Samples should be stored at 4 °C until analysis.
• 15|Identify the best expressed and monodisperse candidate via FSEC (Fig. 3 and Kawate and Gouaux²¹).

Transformation of DH10Bac E. coli (day 5) ● TIMING 1 h

• 16|Transform purified plasmid DNA into DH10Bac E. coli for transposition into the bacmid as described in the Bac-to-Bac system (Invitrogen; http://www.lifetechnologies.com/us/en/home/life-science/protein-expression-and-analysis/protein-expression/insect-expression/bac-to-bac-baculovirus-expression-system.html).

Inoculation of bacmid containing cultures (day 7) ● TIMING 15 min

• 17|Inoculate 5 ml of LB media containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline with a white colony and grow cells overnight at 37 °C.

Isolation of bacmid (day 8) ● TIMING 1 h

• 18|Centrifuge the cells for 10 min at 1,500xg, RT.
• 19|Optional: Make a glycerol stock of the DH10Bac E. coli containing the bacmid DNA for future bacmid DNA isolation. In an autoclaved 1.5 ml centrifuge tube, take 250 μl of cell suspension (from Step 17) and add 250 μl of sterile 50 % glycerol, mix and store for years at −80 °C.
• 20|Discard supernatant and resuspend the pellet in 200 μl of P1 (Qiagen kits). Transfer suspension into a 1.5 ml centrifuge tube.
• 21|Add 200 μl of P2 (Qiagen kits) and mix by inverting the centrifuge tube a few times.

  ! CAUTION Do not vortex samples as this could shear bacmid DNA.

• 22|Add 200 μl of N3 (Qiagen kits) and mix by inverting the centrifuge tube a few times; centrifuge the tube for 10 min at 1,500xg, RT.
• 23|Transfer the supernatant to a 2 ml centrifuge tube and add 1ml isopropanol and gently invert.
• 24|Place the tube for 10 min into −20 °C freezer.
• 25|Centrifuge the tube at 1,500xg for 15 min, RT.
• 26|Remove supernatant, preserve the pellet. Add 1 ml of 70% EtOH and wash the pellet by gently inverting the centrifuge tube.
• 27|Centrifuge the tube at 1,500xg for 15 min, RT.
• 28|Remove supernatant and dry the pellet 5 min.
• 29|Resuspend the pellet in 50 μl of autoclaved MilliQ water. Determine the concentration of the bacmid DNA.

  ! CAUTION Do not pipet samples more than 1–2 times as this could shear bacmid DNA.

  PAUSE POINT Store the bacmid DNA at 4 °C until ready to proceed with Step 30 (up to 3 days).

Transfection of Sf9 cells with bacmid ● TIMING 2 h

• 30|Seed 9×10⁵ of Sf9 cells in 2 ml of Sf-900 media per well of a 6-well plate.

  ? TROUBLESHOOTING

• 31|Incubate cells at 27 °C until they attach (about 20 min).
• 32|while waiting for the cells to attach, add 8 μl of Cellfectin II to 100 μl of Sf-900 III SFM media in centrifuge tubes for each transfection.
• 33|In a different centrifuge tube, add 1 μg of bacmid DNA to 100 μl of Sf-900 III SFM.
• 34|Mix the Cellfectin II/Sf-900 III SFM media mixture and the bacmid DNA/Sf-900 III SFM mixture and incubate for 30 min at RT.
• 35|Change media in each well with 2 ml of Sf-900 III SFM media and add the Cellfectin II/DNA mixture drop wise onto the Sf9 cells. Ensure even dispersal.
• 36|Incubate the cells for 72 hours in 27 °C incubator (make sure to have water inside the incubator to prevent strong evaporation of media).
• 37|Collect supernatant containing P1 virus (~2 ml from each well) and filter the media containing P1 virus into 2 ml centrifuge tube using 3 ml syringe fitted with a small 0.2 μm filter. This is a stock of P1 virus that should be stored at 4 °C light protected for up to a month. Add 2% FBS to stabilize virus stock. It might also be helpful to use the TIPS method⁶⁷ to preserve Sf9 cells infected with P1 virus.
• 38|Determine the titer of the P1 BacMam virus using the Sf9 Easy Titer cell line and the endpoint dilution assay or by using the Virus counter 2100.

  ? TROUBLESHOOTING

Infection of Sf9 cells with P1 virus to produce P2 virus ● TIMING 2 h

• 39|Based on the desired volume of P2 virus, add P1 virus to a MOI of 0.1 to 0.0001 to Sf9 cells that are 1.0–1.5×10⁶ cells/ml in an Erlenmeyer flask of the corresponding size. CRITICAL STEP For the amplification of some viruses we have found that it is essential to infect at a lower MOI than recommended by the Bac-to-Bac system (Invitrogen). Therefore it may be important to determine the optimal MOI for the virus amplification prior to making a large amount of P2 virus.

  ? TROUBLESHOOTING

• 40|Incubate the Sf9 cells infected with the P1 virus for 96 hours in 27 °C orbital shaker at 115 rpm. CRITICAL STEP For the amplification of some viruses the harvest time of the P2 BacMam virus may need to be optimized. We advise initially trying 72 h and 96 h.
• 41|Centrifuge the cells at 8,000xg for 15 min, 4 °C and collect supernatant containing P2 virus.
• 42|Filter the supernatant using disposable 0.2 μm filters (50 ml Steriflip filters from Millipore for small amounts or 250 ml, 0.5 liter, or 1 liter Corning filter systems for large amounts). Add 2% FBS to stabilize virus stock. This is a stock of P2 virus that should be stored at 4 °C light protected (we use aluminum foil) for up to a month.
• 43|Determine the titer of the P2 BacMam virus using the Sf9 Easy Titer cell line and the endpoint dilution assay or by using the Virus counter 2100.

  ? TROUBLESHOOTING

Transduction of suspension HEK293S GnTI⁻ cells with BacMam Virus (day 15) ● TIMING 2 h

• 44|Expansion of HEK293S GnTI- cells should be prepared in advance (~ 10 days in advance) so that a sufficient amount of cells are available on day 15. To expand HEK293S GnTI- cells, determine the total number of cells and percent viability using a hemocytometer and Trypan Blue exclusion and make sure the density of the cells are 2.5 – 3 × 10⁶ cells/ml (from Box 1).
• 45|When a 25 ml culture of HEK293S GnTI⁻ cells are 2.5 – 3 × 10⁶ cells/ml, dilute the culture to 0.2 × 10⁶ cells/ml in 200 ml and incubate the cells on an orbital shaker within a 37 °C incubator in the presence of 8% CO₂ for ~ 5 days until the density is 3 × 10⁶ cells/ml.
• 46|Based on the volume of cells needed (2.4 to 6.4 liters) calculate the volume of media that you need to add to dilute the culture to a seeding density of 0.2 × 10⁶ cells/ml. We prefer to have a starting density of 0.2 × 10⁶ cells/ml. For 2.4 liters one will need 4.8 × 10⁸ cells, approximately 2.2 liters of medium and three 2 liter flasks.
• 47|Aseptically add the appropriate volume of pre-warmed growth medium into the culture flask (the total volume should be 800 ml in a 2L flask). Split the culture to multiple flasks as needed and incubate the cells on an orbital shaker within a 37 °C incubator in the presence of 8% CO₂ for ~ 5–6 days until the cells reach a density of 2–3.5 × 10⁶ cells/ml.
• 48|Add BacMam P2 virus at a MOI of 1 to infect 2.4 liters of HEK293S GnTI⁻ cells at a density of 2–3.5 × 10⁶ cells/ml and incubate the cells on an orbital shaker within a 37 °C incubator in the presence of 8% CO₂.

  CRITICAL STEP The amount of virus added should not exceed more than 10% of the culture volume.

• 49|After 8–24 h at 37 °C add 10 mM sodium butyrate and incubate the cells on an orbital shaker within a 30 °C incubator in the presence of 8% CO₂.

  CRITICAL STEP The incubation temperature for the BacMam transduced HEK293S GnTI⁻ cells should be determined before attempting a large scale expression (Fig. 4). Also the amount and time of sodium butyrate addition should be optimized for each protein (Fig. 5).

• 50|Harvest the cells 60–90 h post transduction by centrifugation for 20 min at 6,200xg, 4 °C.

  CRITICAL STEP The harvest time of BacMam transduced HEK293S GnTI⁻ cells should be determined before attempting a large scale expression (Fig. 4).

  ? TROUBLESHOOTING

  PAUSE POINT The cell pellets can be stored at −80 °C for weeks until ready to proceed with protein purification ^15–17,20.

TROUBLESHOOTING

Troubleshooting advice can be found in Table 1.

TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
1, 30, 39, 43, Boxes 1 and 2	Change in media color and/or presence of foul smell	Contamination of cell culture due to improper sterile technique	Ensure proper sterile technique is used. Clean and disinfect hood work surfaces before and after every manipulation. Before working in the hood disinfect gloves with 70% alcohol. Spray all items to be placed in the hood (media bottles, pipetting devices, outside of the plastic wrap the sterile containers, etc) with 70% ethanol and wipe them with paper towels.
		Contaminated virus	Ensure virus has been filter sterilized.
		Contaminated reagent	Filter sterilize histone deacetylase inhibitors (i.e. sodium butyrate) in the hood.
38	Low virus titer	Sf9 cells infected at the wrong MOI	Generate P2 virus using lower MOI (0.05–0.0001).
			Remake the bacmid ensuring that the bacmid culture is inoculated from a white colony and that the bacmid DNA is not sheared during purification.
45, 50	Low cell harvest density	Poor-quality cells	It is important not to let cells overgrow (i.e. to subculture them on a regular basis) and to avoid using cells that have undergone more than 30 continuous passages since being raised from liquid nitrogen. Check health and viability of cells prior to each experiment.
	Low expression	Infection at the wrong MOI (i.e. too little or too much virus), non-optimal expression conditions, toxicity of membrane protein when overexpressed	Always determine the titer of the BacMam virus to caculate a MOI. Screen for optimal temperature, harvest time and concentration of histone deacetylase inhibitors.
		Virus is not stable when stored for extended periods	Remake the P2 virus starting from either the P1 virus or a glycerol stock of DH10Bac E. coli containing bacmid DNA. Use TIPS method66 to preserve Sf9 cells infected with P1 virus.

● TIMING

The entire protocol, starting from transfection (Step 1) to the harvest of BacMam virus transduced suspension HEK293S GnTI⁻ cells (Step 50), takes approximately 3 weeks to complete if a promising candidate is identified (Step 14). The hands-on timing for each stage of the procedure is summarized below.

• Step 1, Cell seeding: 15 min

• Steps 2–7, Small scale transient transfection to screen constructs: 45 min

• Steps 8–15, Screen constructs by FSEC for monodispersity and expression level: 3 h

• Step 16, Transformation of DH10Bac E. coli: 1 h

• Step 17, Inoculation of bacmid containing cultures: 15 min

• Steps 18–29, Isolation of bacmid: 1 h

• Steps 30–38, Transfection of Sf9 cells with bacmid (producing P1 virus): 2 h (45 min transfection + 15 min P1 virus harvest + 1 h virus titer determination using the Virus Counter 2100)

• Steps 39–43, Infection of Sf9 cells with P1 virus to produce P2 virus: 2 h (15 min infection of Sf9 cells with P1 virus+ 45 min harvest P2 virus + 1 h virus titer determination using the Virus Counter 2100)

• Steps 44–50, Expansion of HEK293S GnTI⁻ cells and transduction of suspension HEK293S GnTI⁻ cells with BacMam Virus: 2 h

• Box 1, Growth and maintenance of suspension HEK293S GnTI⁻ cells: 15 min

• Box 2, End point dilution assay: 1 h

ANTICIPATED RESULTS

This protocol (as outlined in Fig. 1) has been used in our laboratory to successfully express cASIC1, Drosophila melanogaster dopamine transporter (DAT), N-methyl-D-aspartate (NMDA) receptors and many other membrane proteins in HEK293S GnTI⁻ cells^15,16,20,61. The time it takes to identify a promising candidate (Fig. 3), is likely to vary significantly depending on (for example) the number of flexible regions to be removed, surface entropy reduction mutations and thermostability mutations. Although few changes made for cASIC, several construct changes were needed for DAT and NMDA to obtain the best expressed and monodisperse candidate via FSEC^15–17,20. Once a promising candidate is identified, the most favorable conditions for MOI, cell density, expression time, temperature (Fig. 4), and the presence of histone deacetylase inhibitors (i.e. sodium butyrate; Fig. 5) should be determined for each protein before attempting a large scale expression. The protocol for optimized expression can be completed in approximately 3 weeks. Purification of membrane proteins from transduced HEK293S GnTI⁻ cell pellets (which may include Affinity Chromatography, tag cleavage, removal of N-linked glycosylation, and size exclusion chromatography), depending on the candidate protein, can produce 0.25–1.5 milligrams of protein per liter of media sufficient for crystallization. The protein expression and yield can vary depending on factors such as the titer of the virus, the toxicity of the protein when expressed, and the stability of the protein.