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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Protoc. Author manuscript; available in PMC 2013 April 1.
Published in final edited form as:
PMCID: PMC3613139
NIHMSID: NIHMS428713

Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells

Abstract

X-ray crystal structures of human membrane proteins, while potentially being of extremely high impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we utilize to overexpress human membrane proteins from clonal HEK293S GnTI- cells, and was recently used in our 2.1 Å X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled-up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ~0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost-effective, can be used to express proteins that would otherwise be toxic to mammalian cells, and can be completed in 8–10 weeks.

Introduction

The overexpression of human membrane proteins, in both quantities and qualities amenable for structural studies, is often an immense challenge for numerous reasons. At a fundamental level, the biosynthesis of both prokaryotic and eukaryotic membrane proteins requires two events to occur1,2. Firstly, the newly synthesized polypeptide chain must be inserted into the membrane via recognition by the evolutionarily conserved Sec translocon3,4. This prerequisite for Sec translocon engagement can be seen as placing a lower ceiling on membrane protein biosynthesis, relative to soluble protein biosynthesis, given that the Sec translocon can become saturated when membrane proteins are overexpressed5,6. The manner by which the Sec translocon can recognize heterologous sequences can lower this ceiling even further7, in particular in those cases of expressing human membrane proteins in the most commonly used system for protein expression, Escherichia coli8. Secondly, the inserted polypeptide must laterally traverse into the lipid bilayer where folding into the correct three-dimensional structure predominately occurs1,2,9. Given that the composition of lipid bilayers differ substantially between humans and other species10,11, and given the pronounced effects that lipids and sterols have on membrane protein structure and function1214, it is apparent that lipid composition is a key variable in determining the yields of functional membrane proteins that are produced.

In addition to these two fundamental principles of membrane protein biosynthesis, eukaryotic membrane proteins often require additional post-translational modifications not found in prokaryotes. N-linked glycosylation of eukaryotic membrane proteins, for instance, can be of critical importance for the folding of membrane proteins possessing large extracellular domains, given that these glycans serve as ligands for the ER-resident molecular chaperones calnexin and calreticulin15. Nevertheless, N-linked glycosylation is often seen as an impediment towards structure determination via X-ray crystallography, given that these glycans are often very large, heterogeneous, and conformationally flexible16. Indeed, considerable effort has been placed into restricting the heterogeneity of these N-linked glycans, via mutagenesis, glycosidases and/or inhibitors, for the purpose of structural studies1720.

The use of mammalian cell expression systems is becoming increasingly popular for the overexpression of mammalian membrane proteins for structural studies21,22, likely owing to their near-native translocation machinery, lipid milieu, and post-translational modifications. A suspension adapted Human Embryonic Kidney 293 (HEK293S) cell line lacking N-acetylglucosaminyltransferase I (GnTI) has been successfully employed to overexpress a wide variety of mammalian membrane proteins2325, including that of human RhCG whose X-ray crystal structure was recently reported in our laboratory21. Developed by Gobind Khorana and colleagues to overexpress large quantities of functional rhodopsin20, the HEK293S GnTI- system possesses features that make it amenable for structural studies. Firstly the lack of GnTI restricts N-linked glycans to a homogeneous Man5-GlcNac2 structure, which greatly facilitates their enzymatic removal via endo- and exoglycosidases. Secondly, the system utilizes a tetracyclin inducible promoter, allowing for protein expression to be induced once high-density cell cultures are established (discussed further in Induction of membrane protein expression). As compared to other lower eukaryotic expression systems that have been used to express mammalian membrane proteins for structural studies, such as baculovirus infected SF9 insect cells26, expression from HEK293S GnTI- cells is typically more labor intensive and less cost-effective. Nevertheless, given that the folding and proper trafficking of mammalian membrane proteins can depend critically on the translocation machinery, lipid milieu, and post-translational modification present in the expression system14, the use of mammalian cell expression systems likely ensures the greatest probability of producing properly folded and functional human membrane proteins.

In this protocol, we describe the methods that we used to overexpress quantities of pure, homogeneous and stable human RhCG from stably transfected and clonal HEK293S GnTI-cells, for use in crystallization. Expression trials of human RhCG were performed using Escherichia coli, Saccharomyces cerevisiae, and HEK293S GnTI- cells, however, only in the case of HEK293S GnTI- cells was RhCG expressed at high levels sufficient for structural studies. In addition, three human Rh proteins were transiently expressed in HEK293S GnTI- cells, with RhCG possessing the highest level of solubilized membrane protein (Figure 1). The protocol described here is of use to those that require long-term overexpression of a human membrane protein, for example in antigen production, drug discovery, biochemical characterization, or structural studies, and assumes that suitable expression constructs have already been generated and screened (discussed further in Experimental Design). A summary of the various steps of the protocol are discussed in detail below.

Figure 1
Expression testing of full-length human Rh membrane proteins in transiently transfected HEK293S GnTI- cells. 10 μg of RhAG, RhBG and RhCG, subcloned into pACMV-tetO, were separately transfected into HEK293S GnTI- cells grown in 10 cm2 tissue culture ...

Stable versus transient expression in HEK293S cells

There are two ways by which a transfected transgene can be overexpressed in HEK293S cells, either transiently or stably. In transient expression, a sufficient quantity of plasmid DNA is transfected into cell cultures of varying sizes (mL to L scales), after which overexpression is either immediate or induced, dependent on the promoter type of the plasmid. Continued passage of transiently transfected cells results in a dilution of cells that are transfected, therefore, each overexpression trial must be preceded by transfection. Large scale transient expression, typically involving cell cultures in the 1–10 L range, is becoming increasingly popular given the flexibility of the system and given its potential for high-throughput overexpression trials17,19,27.

Stable expression, on the other hand, occurs from a transgene that has been stably integrated into the transfected cell’s genome. Stably transfected cell lines are generated by introducing Geneticin to the cell culture, over a period of 2–3 weeks, which selects for cells that possess stably integrated plasmid DNA (possessing the transgene and Geneticin resistance). Clones of stably transfected cell lines can then be generated and scaled up for transgene overexpression. The generation of clonal, stably transfected HEK293S cell lines is initially slower and more technically challenging relative to large scale transient expression. Nevertheless, once a clonal cell line is generated, long-term overexpression from stably transfected cells can be much more facile and consistent compared to transient expression given that the purification of large quantities of plasmid DNA, followed by separate large-scale transfections, is not required.

We have explored overexpression of human RhCG from both transiently and stably transfected HEK293S cell lines, using a pACMV-tetO expression vector in both cases, and have found that expression levels are significantly higher in clonal, stably transfected HEK293S cells. Nevertheless, in those cases where shorter time-scales and/or higher throughput are favored over longer-term and consistent expression levels, cell lines and expression vectors designed for transient expression can be explored17,28.

Stable cell line generation

Stably transfected HEK293S cells are generated by selecting for successful integration of the neo gene using the antibiotic Geneticin, following a workflow as shown in Figure 2. Selection occurs in a 10 cm2 tissue culture plate, where it takes ~2–3 weeks for foci of Geneticin resistant colonies to appear, after which the resistant foci are clonally expanded over a period of an additional ~2–3 weeks. While we used Geneticin for selection, other drugs and resistance gene combinations can be used, for instance puromycin and puromycin acetyltransferase, respectively.

Figure 2
Workflow for the overexpression of mammalian membrane proteins in stably transfected and clonal HEK293S GnTI- cells.

The level of transgene expression for any given stably transfected, and clonal HEK293S cell line can vary substantially. Integration of plasmid DNA into the HEK293S genome is random, therefore, the level of transgene expression will be determined in large part by position effects. In addition, given that the site of recombination within the plasmid sequence is random, no transgene expression will be observed if the site of recombination disrupts either the coding sequence or the promoter of the transgene. We have found that expanding 24 clonal cell lines, and screening these clones for expression via western blotting (discussed below in Assessment of membrane protein expression levels from clonal cell lines), is most efficient to find the highest expressing clones. Nevertheless, in those cases where screening 24 clonal cell lines is insufficient to identify highly expressing clones, or in those cases where a large number of clonal cell lines are to be generated in parallel, higher throughput cloning and screening strategies could be explored. For example, higher throughput cloning into 96 well plates, via fluorescent-activated cell sorting (FACS) or limited dilution plating, followed by fluorescence or ELISA quantification of expression levels, can be used to facilitate cloning and screening for highly expressing mammalian cell lines29.

Assessment of membrane protein expression levels from clonal cell lines

Small scale detergent solubilizations, using both β-octylglucoside (OG) and β-dodecylmaltoside (DDM), are performed on all clonally expanded cell lines. The use of two or more detergents at this stage is optional, as detergent solubilization screening can be performed further downstream upon identification of the highest expressing clone. DDM should be used for small scale solubilizations if a single detergent is to be assessed, given its greater ability to solubilize membrane proteins compared to OG30.

The solubilized material from each clonal cell line is assessed for membrane protein expression via anti-fusion tag western blotting. Comparison of the “before spin” (BS) sample, which reveals total membrane protein expression levels, with the “after spin” (AS) sample, which reveals successfully solubilized protein expression levels, indicates the degree of solubilization. Caution should be exercised in those cases where solubilization only occurs in fos-choline type detergents, given their potential to denature membrane proteins31. Although the western blotting protocol used for assessing membrane protein expression levels is not quantitative, relative expression levels between clones can be assessed, allowing for the identification of clones that express high levels of detergent-soluble membrane proteins. It is the amount of detergent-soluble membrane protein (i.e. AS) produced, and not the total amount of membrane protein (i.e. BS) produced, that defines the useable expression level of a particular cell line.

Medium and large scale suspension cultures

Once a highly expressing, clonal cell line has been identified, medium (~1–3 L spinner flask) and/or large (~10 L cellbag bioreactor) suspension cultures are established for overexpression trials. The HEK293S GnTI- cells used during the course of this work are adaptable for suspension growth20, allowing for the establishment of higher densities of cell cultures relative to adherent cultures. This adaptation from an adherent monolayer to suspension cultures is brought about solely by changing the medium; the suspension medium lacks the calcium required for forming an adherent monolayer. In practice, adherent HEK293S GnTI- cells do not take long to be adapted to grow as a suspension culture in DMEM supplemented with serum20,32. Nevertheless, the growth of HEK293 cells in suspension is not always facile and, dependent on the type of cells and media used, can require that the cells be adapted for suspension growth over a period of several weeks28. The suspension medium used in this manuscript is a cost effective alternative to commercially available, serum-free formulations designed for suspension cell cultures and, in addition to a lack of calcium, includes Iron-supplemented Bovine Calf Serum (BCS), and the non-ionic detergent Pluronic F-68, with Primatone RL/UF added later as a supplement. Iron-supplemented BCS is a cost effective alternative to Fetal Bovine Serum (FBS), while Primatone RL/UF increases the viability and density of mammalian cells grown in suspension33. Pluorinic F-68 has been shown to both reduce cell adherence and to protect cells against high levels of shear stress introduced by sparging and stirring in the suspension culture34, in a mechanism thought to involve the non-ionic detergent coating the plasma membranes of the suspension cultures34. This suspension medium permits us to grow HEK293S GnTI- cells as a suspension culture, without requiring a lengthy adaptation period from an adherent culture.

The scale of cell culture that is established depends largely on what is known of the membrane protein being expressed. At the initial stages of the structure determination of human RhCG, 1–2 L spinner flasks were grown, induced, and harvested on a periodic basis. These 1–2 L spinner flask cultures were used to determine the optimal parameters for isolating pure, homogeneous and stable RhCG; parameters which included detergent type, pH, glycerol and protease concentrations and incubation periods (for fusion tag removal). Once optimal purification conditions were determined, larger 10 L cellbag, or occasionally 3 × 3 L spinner flask, suspension cultures were established. While cellbag bioreactors are expected to establish higher densities of suspension cultures relative to spinner flasks, owing to more efficient oxygen transfer into the medium, the observed densities of RhCG HEK293S cells in cellbag and spinner flask cultures were comparable (~1–1.5 × 106 cells/mL). We have not, however, explored if increasing or decreasing the rate of air/oxygen sparging into the cellbag can increase cell densities. Given the comparable cell densities observed for RhCG HEK293S in cellbags and spinner flasks, we have also established large scale (3 × 3L) spinner flask cultures of RhCG HEK293S, which are more facile and more cost efficient compared to cellbag cell cultures.

Induction of membrane protein expression

In the HEK293S GnTI- system all membrane proteins are expressed from a tetracycline-inducible promoter. This is an advantage in the overexpression of membrane proteins in particular, since the intrinsic function of certain membrane proteins (e.g. channels, GPCRs, etc.) can be cytotoxic to the cell, thereby preventing high density cell cultures from being established14,32,35,36. For example, it has been shown that stable cell lines constitutively expressing the serotonin transporter (SERT) can only be generated in the presence of SERT inhibitors, possibly owing to the fact that the intrinsic functions of SERT (serotonin transport and channel-like activity) severely stress the cell, in the absence of SERT inhibitors14. The use of a tetracycline-inducible promoter, therefore, delays the expression of potentially toxic membrane proteins until high-density cell cultures are established.

In the protocol described here, membrane protein expression is induced by adding an appropriate amount of doxycycline (a tetracycline antibiotic) to the suspension culture, either grown in spinner flasks or cellbags. For RhCG, we have found that induction with doxycycline at a cell density of ~1.0 × 106 cells/mL, followed by harvesting the cells 36 hours later, is optimal for RhCG expression, nevertheless, these parameters should be empirically determined for each particular membrane protein to be overexpressed. Sodium butyrate is also added to the suspension cultures at the time of induction, given its ability to increase protein expression levels from mammalian cells37, however it alone does not induce expression.

Whole cell solubilization and membrane protein purification

Solubilization of RhCG from whole HEK293S cells was performed subsequent to harvesting the medium- and large-scale suspension cultures. We have found that whole cell solubilizations are faster to perform and can result in higher yields of extracted RhCG compared with solubilization from a membrane preparation. Solubilization of membrane proteins from a membrane preparation, however, should be explored if the final purity of membrane protein after whole cell solubilization is insufficient.

A two-step purification scheme was utilized for the purification of human RhCG. Firstly, FLAG purification was performed, with either OG and DDM, from whole cell solubilized RhCG. This step will depend on the type of transgene that is integrated into the clonal cell; in the case of RhCG, the expression vector (pACMV-tetO20) was modified to possess an N-terminal FLAG and a C-terminal His tag, potentially allowing for two types of affinity purification to be performed. The FLAG purified RhCG was more pure and of better yield, as compared to the immobilized metal affinity chromatography (IMAC) purified RhCG, therefore we did not explore IMAC purification in detail. Secondly, size exclusion chromatography was performed, which resulted in the purification of RhCG to near homogeneity. Size exclusion chromatography is an excellent metric for both the purity of a membrane protein (pure proteins will possess discrete and near-monodisperse major peaks) and its stability (stable proteins will possess few changes in chromatographic profile over multiple days). Purified membrane proteins that are both near-monodisperse and stable, such as was found with human RhCG, are excellent candidates for crystallization trials.

Experimental Design

Prior to undertaking the experiments described in this protocol, it is important to screen potential constructs for both expression quality and quantity. The use of fluorescence size exclusion chromatography (FSEC) at this stage to assess protein quality and quantity from transiently transfected HEK293 cells is therefore suggested, in that this will help to ensure that only suitable expression constructs are selected for stable cell line generation38. Constructs suitable for structural studies will possess single, monodisperse FSEC profiles of high magnitude38. An alternative approach is to perform anti-fusion tag western blotting of transiently transfected HEK293S GnTI- cells, in a similar manner as described in this protocol (see steps 26–34). Constructs that possess weak signal on a western blot could then be redesigned, or possibly codon optimized, depending on the importance of the particular membrane protein to the laboratory. The screening of constructs via FSEC or western blotting is also important in that, in our experience, it is not possible to predict a priori if a particular membrane protein class will express well in HEK293S GnTI- cells, as the highest expressing clonal cell lines produced in our laboratory, to date, are an ammonia transporter (RhCG), a cation transporter, a class B GPCR, and a protease.

While the entire procedure described here takes approximately 8–10 weeks to perform, the procedure can be paused at week 6 by freezing the clonal cell line. It is important to ensure that personnel are available to maintain the HEK293S GnTI- cells up until week 6, as clonal cell line generation can fail if the procedure is not followed and the cells are unnecessarily stressed (e.g. by failing to change the medium promptly, allowing the cells to become over confluent, etc.). This requires approximately 0.5–3 hours of cell culture work per day, 1–4 days per week, depending on the stage of the procedure. Similarly, once HEK293S GnTI- cells are harvested from spinner flask or WAVE cellbag suspension cultures, the whole cell membrane solubilization and purification steps should be performed immediately, given that the membrane protein of interest may be unstable; this most closely follows the procedure performed for the solubilization, purification and crystallization of human RhCG. Nevertheless, the procedure can be modified following the cell harvest stage to create an additional pause point by preparing a membrane fraction and freezing these membranes at −80°C39, for future solubilization and purification.

MATERIALS

REAGENTS

  • HEK 293S GnTI- cells (ATCC# CRL-3022)
  • pACMV-tetO (See Supplementary Figure 1 for full sequence)
  • Opti-MEM I (UCSF Cell Culture Facility, cat. no. CCFAC008 or Invitrogen, cat. no. 31985070) ▲CRITICAL All reagents to be used for cell culture work (media, antibiotics, chemicals, etc.) should be of tissue culture grade, if available.
  • Dulbecco’s Modified Eagle Medium (DMEM) high glucose (UCSF Cell Culture Facility, cat. no. CCFAA005 or Invitrogen, cat. no. 32430027)
  • Dulbecco’s Modified Eagle Medium (DMEM) high glucose without Ca salts (UCSF Cell Culture Facility, cat. No. CCFDA003 or Invitrogen, cat. no. 21068028)
  • D-PBS (without calcium and magnesium) (UCSF Cell Culture Facility, cat. no. CCFAL003 or Invitrogen, cat. no. A1285601)
  • Penicillin- Streptomycin (100x) (UCSF Cell Culture Facility, cat. no. CCFGK004 or Invitrogen, cat. no. P4333-100ML)
  • Sodium bicarbonate (Sigma, cat. no. S5761)
  • Trypsin (0.05% w/v trypsin with EDTA in Saline A) (UCSF Cell Culture Facility, cat. no. CCFGP002 or Invitrogen, cat.no. 25300-054)
  • Geneticin (50mg/ml) (Gibco, cat. no. 10131-027)
  • Blasticidin S HCl (50mg) (Invitrogen, cat. no. R210-01)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668-019)
  • Pluronic (Sigma, cat. no. P1300)
  • Sodium butyrate (Sigma, cat.no. 303410)
  • Doxycycline hyclate (Sigma, cat.no. D9891)
  • Primatone RL/UF (Kerry Bio Science, cat. no. 5X00130)
  • Foetal bovine serum (FBS) (JRScientific Inc., cat. no. 43652)
  • Cell preservation media (UCSF Cell Culture Facility, cat. no. CCFHH002)
  • Iron-Supplemented Bovine Calf Serum (BCS) (Hyclone, cat no. SH30072.03)
  • Tris Base (Fisher Scientific, cat. no. BP154-1)
  • NaCl (Fisher Scientific, cat. no. 7647-14-5)
  • Glycerol (Fisher Scientific, cat. no. BP229-4)
  • ANTI-FLAG M2 Affinity Gel (Sigma, cat. no. A2220)
  • 3X FLAG peptide (Sigma, cat. no. F4799)
  • Octyl-β-D-glucopyranoside (OG) (Anatrace, cat. no. O 311)
  • N-dodecyl- β-D-maltopyranoside (DDM) (Anatrace, cat. no. D 310)
  • Phenylmethanesulfonyl fluoride (PMSF) (Sigma, cat. no. P7626)
  • Complete EDTA-free Protease Inhibitor Cocktail Tablets (Roche, cat. no. 11 873 580 001)
  • Western blot reagents (Thermo, cat. no. 1856135 and 1856136)
  • Benzonase (Sigma, cat. no E1014)
  • Dithiothreitol (Sigma, cat. no. D0632)
  • 3C protease (homemade)
  • Thrombin (Novagen, cat. no. 69671-3)
  • Mouse IgG-Agarose (Sigma, cat. no A0919)

EQUIPMENT

  • CryoTube vials (Nunc, cat. no. 377267)
  • Cloning cylinder (Bel-Art products, cat. no. 378470100, 378470200 and 378470300)
  • Tissue culture plate, 6 well (BD Falcon, cat. no. 353046)
  • Tissue culture plate, 24 well (BD Falcon, cat. no. 353047)
  • Tissue culture dish, 100mm (BD Falcon, 353003)
  • Tissue culture dish, 150mm (BD Falcon, 353025)
  • Sterile alcohol prep pads (Fisher HealthCare, 06-669-62)
  • Precast 4–20% w/v SDS-PAGE (Biorad, cat. no. 456-1096)
  • 7 mL, 15 mL, and 40 mL Dounce homogenizers (Kontes Glass Co.)
  • 1000 mL bottle (Beckman)
  • 250 mL bottle (Nalgene)
  • PCR machine (Biorad or equivalent)
  • Inverted light microscope (Olympus CK2 or equivalent)
  • Class II biological safety cabinet (The Baker Company)
  • CO2 Incubator (Sanyo)
  • Superdex 10/300GL (GE Healthcare)
  • Fast performance liquid chromatography (FPLC) station (Akta, Shimadzu, or equivalent)
  • AHB Spin Flask Comp (1 L and 3L (Bellco) ▲CRITICAL Ensure that each flask has been cleaned and autoclaved prior to use (see Equipment setup)
  • Wave Bioreactor (GE Healthcare)
  • Cell bag (GE Heaththcare or Flex Concepts)
  • Nutator (BD)
  • Ultrasonic water bath (Bransonic)
  • mosquito® (TTP LabTech)
  • Optima L-90K Ultracentrifuge (Beckman)
  • Type 45 Ti rotor (Beckman)
  • Glass Econo-column (Biorad, cat. no. 737-1517)
  • 5 mL luer syringe (BD)
  • 0.22 and 0.45 μm syringe filters (Pall)
  • 15 mL and 50 mL centrifuge tubes (Corning)
  • 7 mL, 15 mL, and 40 mL Dounce homogenizers (Kontes Glass Co.)
  • Amicon® Ultra-15 (50K or 100K cutoff) Centrifugal Filters (Millipore, cat. no. UFC905024 and UFC910024)

REAGENT SET UP

DMEM medium

To 1 L of DMEM high glucose, add 10 mL of Penicillin- Streptomycin (100X) and 100 mL of Iron-Supplemented BCS. Filter-sterilize inside a biological safety cabinet using a 0.2 μm syringe filter. Store at 4°C for up to 1 year. ▲CRITICAL The addition of serum to DMEM is suggested for all cell culture work as, in our experience, HEK293S GnTI- cells do not passage well in DMEM alone. Iron-Supplemented BCS is added to both DMEM and Suspension medium, as it is a cost-effective alternative to FBS, suitable for most cell culture work (see DMEM (10% v/v FBS)for exception).

DMEM (10% v/v FBS)

To 1 L of DMEM high glucose, add 10 mL of Penicillin- Streptomycin (100X) and 100 mL of FBS. Filter-sterilize inside a biological safety cabinet using a 0.2 μm syringe filter. Store at 4°C for up to 1 month. ▲CRITICAL Medium that is used during the drug selection process and foci expansion contain FBS, in place of Iron-supplemented BCS, in order to better ensure cell viability.

Selection Medium

To 1 L of DMEM high glucose, add 10 mL of Penicillin- Streptomycin (100X), 100 mL of FBS, 40 mL of Geneticin, and 1 mL of Blasticidin S HCl. Filter-sterilize inside a biological safety cabinet using a 0.2 μm syringe filter. Store at 4°C for up to 1 month. ▲CRITICAL Medium that is used during the drug selection process and foci expansion contain FBS, in place of Iron-supplemented BCS, in order to ensure better cell viability.

Suspension Medium

To 1 L of DMEM high glucose without calcium salts, add 10 mL of Penicillin- Streptomycin (100X), 10 mL of pluronic, 0.3 g of Primatone RL/UF, 100 mL of BCS, and 3.7 g of sodium bicarbonate. Filter-sterilize inside a biological safety cabinet using a 0.2 μm syringe filter. Store at 4°C for up to 1 month. !CAUTION Serum-free mediums formulated for the growth of HEK293S in suspension, such as 293 SFM II (Invitrogen, cat. no. 11686-029), may also be used, however they are typically five-fold more expensive as compared to Suspension Medium.

Suspension Medium (For cellbag bioreactor suspension cultures)

To 7 L of DMEM high glucose without calcium salts, add 70 mL of Penicillin- Streptomycin (100X), 700 mL of BCS, 70 mL of pluronic, 2.1 g Primatone Rl/UF and 25.9 g sodium bicarbonate. Prepare prior to use and store at 4°C.

5 mg/mL Blasticidin solution

Dissolve 50 mg of Blasticidin S HCl in 10 mL of autoclaved water. Filter-sterilize inside a biological safety cabinet using a 0.2 μm syringe filter and store 1 mL aliquots at −20°C. !CAUTION Blasticidin is toxic. Always wear gloves, a mask, a laboratory coat, and safety glasses. Prepare the solution inside a biological safety cabinet. Aliquot in small volumes suitable for one time use, and store at −20°C for up to 6–8 weeks.

20 mg/mL Doxycycline Hyclate

To prepare 20 mg/mL of doxycycline solution, dissolve 100 mg of doxycycline hyclate in 5 mL of autoclaved water. Filter-sterilize inside a biological safety cabinet using a 0.2 μm syringe filter and store 1 mL aliquots at −20°C for up to 6–8 weeks. !CAUTION Doxycycline should not be exposed to direct sunlight.

500 mM Sodium Butyrate

Dissolve 27.5 g of sodium butyrate with autoclaved water to a final volume of 500 mL and filter-sterilize using 0.2 μm syringe filter inside the biological safety cabinet. Store at room temperature (20–25°C) for up to 6–8 weeks.

10% w/v Primatone RL/UF

Dissolve 3g of Primatone RL/UF in 30 mL of autoclaved water and filter-sterilize inside the biological safety cabinet using a 0.2μm syringe filter inside the biological safety cabinet. Prepare immediately before use.

20% w/v Glucose

Dissolve 100 mL of glucose in 500 mL of autoclaved water and filter-sterilize inside the biological safety cabinet using a 0.2μm syringe filter inside the biological safety cabinet. Store at 4°C for up to 2–3 months.

10% w/v Pluronic

Dissolve 50 mL of pluronic in 500 mL of autoclaved water and filter-sterilize using a 0.2μm filter inside the biological safety cabinet. Store at 4°C for up to 6–8 weeks.

2X Western solubilization buffer

40 mM Tris pH 7.4, 200 mM NaCl, 20% v/v glycerol. Immediately before use, add 1 mM PMSF and one complete EDTA-free protease inhibitor cocktail tablet. Chill to 4°C, and discard any unused buffer.

Lysis buffer

20 mM Tris pH 7.4, 100 mM NaCl, 10% v/v glycerol. Immediately before use, add 1 mM PMSF and one complete EDTA-free protease inhibitor cocktail tablet. Chill to 4°C, and discard any unused buffer.

Size-exclusion chromatography (SEC) buffer

20 mM Tris pH 7.4, 100 mM NaCl, 10% v/v glycerol, 40 mM OG (or 0.5 mM DDM). Store at 4°C.

Culture of HEK293S GnTI- cells

Periodically split into 10 cm2 tissue culture plates using DMEM. In a biosafety cabinet, gently wash the cells with 5 mL of D-PBS and aspirate off the medium. Add 1 mL of trypsin, and incubate at room temperature for 1 minute. Gently shake the tissue culture plate to facilitate dislodging of the cells from the plate. Add 5 mL of DMEM, resuspend the cells, and pellet at low speed for 5 minutes. Discard the DMEM, add 1 mL of DMEM to resuspend the cells, and add half of the cells to a new 10 cm2 tissue culture plate containing 10 mL of DMEM. For all cell culture work, HEK293S cells are maintained in a humidified incubator set to 5% CO2/95% air and 37°C. ▲CRITICAL STEP HEK293 cells, as with mammalian cells in general, are very easily contaminated. Therefore, it is of utmost importance that sterile tissue culture techniques are followed at all times. All surfaces (i.e. biosafety cabinet, microscopes, etc.) should be cleaned with 70% v/v ethanol prior to use. Gloves (cleaned with 70% v/v ethanol) and lab coats must be worn at all times.

? Troubleshooting

EQUIPMENT SET UP

Spinner flask

Clean thoroughly, by separating the different parts of the flask. Add 10% v/v glacial acetic acid, and stir overnight at room temperature. Next day, remove the 10% v/v glacial acetic acid and rinse the spinner flask very well to remove any trace of acid. Fill the spinner flask with distilled water and perform two liquid autoclaves for 30 minutes each, and then a final dry autoclave for 30 minutes. Allow the flasks to cool to room temperature before use.

WAVE Bioreactor

Assemble the WAVE bioreactor in a tissue culture room, following the manufacturer’s protocols.

PROCEDURE

Day 1: Cell seeding ● TIMING ~15 min

  • 1|
    Split HEK293S cells to ~40% confluency into each well of a 6 well tissue culture dish, using DMEM, so the next day cells are ~70–80% confluent for transfection. In practice, this can be performed by resuspending the cells from one confluent 10 cm2 tissue culture plate with 6 mL of DMEM, and seeding approximately 400 μl of this into a well containing 2 mL of DMEM.
    ? Troubleshooting

Day 2: Transfection ● TIMING ~5–7 hr: 45 min Transfection + 4–6 hr incubation + 15 min media exchange

  • 2|
    On the day of transfection, ensure that the cells are ~70–80% confluent.
    ? Troubleshooting
  • 3|
    In an autoclaved 1.5 mL centrifuge tube, dilute 1.5 μg of plasmid DNA (in up to 10 μL of autoclaved H2O) with 240 μL of Opti-MEM I.
  • 4|
    Repeat step 3 with positive control DNA (i.e. typically a pACMV-tetO construct containing a well expressed transgene) and 10 μL of autoclaved H2O as negative control.
  • 5|
    For each transfection to be performed, dilute 30 μl of Lipofectamine 2000 with 720 μl of Opti-MEM I in a separate 1.5 mL centrifuge tube and incubate for 5 minutes at room temperature.
  • 6|
    Gently mix the diluted DNA samples (steps 3 and 4) with the diluted Lipofectamine reagent (step 5) (total volume = 500 μl), and incubate for 30 minutes at room temperature to allow for the formation of DNA-liposome complexes.
  • 7|
    During the 30 minutes incubation of step 6, replace the medium of the cells with 1.5 mL of Opti-MEM I.
  • 8|
    To one well of the six well cell culture plate, dropwise add 500 μl of DNA-liposome complex. A six well tissue plate can, therefore, be used to transfect four separate constructs, in addition to the positive and negative controls. Mix gently by tilting the plate back and forth and incubate for 4–6 h at 37°C in a CO2 incubator.
  • 9|
    Replace the medium with DMEM and incubate overnight at 37°C.

Day 3: Expansion ● TIMING ~2hrs

  • 10|
    Split the cells from one well of the six well plate into five 10 cm2 tissue culture plates, at a dilution ratio ranging from 1:20 to 1:400, as shown in Figure 3 and incubate overnight at 37°C.
    Figure 3
    Dilution of tranfected HEK293S GnTI- cells prior to drug selection. Cells from one well of a 6 well tissue culture plate (2 mL total) are diluted into five 10 cm2 tissue culture plates, by plating the appropriate amount of cells (indicated in the boxes). ...
    ? Troubleshooting

Day 4: Geneticin selection ● TIMING ~15min

  • 11|
    Exchange the DMEM in each of the 10 cm2 plates with 10 mL of Selection Medium and incubate overnight at 37°C.

Generation of stably transfected cell colonies ● TIMING 2–3 weeks

  • 12|
    Exchange the Selection Medium in each of the 10 cm2 plates with 10 mL of fresh Selection Medium. During the first week of selection, the medium should be replaced every other day, while after one week the medium can be replaced every 3–4 days. The whole process takes approximately 15–20 days, after which single colonies of adequate size for clonal expansion (~1 cm in diameter) are formed.
    ? Troubleshooting

Generation of clonal cell lines ● TIMING 2–3 weeks

  • 13|
    Using a felt tip pen, draw a circle on the back of the tissue culture plate around up to 24 single, well isolated cell colonies. Hold the plate towards a light source for better colony visualization. ▲CRITICAL STEP Colonies must be single and well isolated from neighboring colonies, to ensure that clonal cell lines are generated. Of the five cell dilutions prepared in step 10, only a few well isolated colonies are typically observed for the most diluted plate (1:400), while less well isolated colonies are observed for the least diluted (1:20) plate. Choose a total of 24 colonies from all of the five 10 cm2 plates.
  • 14|
    Based on the size of the colony, choose either a small, medium, or large sized cloning cylinder. Use sterile forceps to pick up the sterile cylinder, and apply autoclaved grease to the bottom of the cylinder, to ensure that the cylinder will seal to the plate.
  • 15|
    Aspirate off the medium from the plate and wash the cells with 5 mL of D-PBS. Carefully, without disturbing the colony or touching any other colony, place a cylinder over the colony such that the colony is centered in the middle of the cylinder. Gently press the cylinder down to adhere it to surface. ▲CRITICAL STEP Make sure to completely aspirate off the D-PBS, as any remaining D-PBS will prevent the cylinder from adhering well to the plate.
  • 16|
    Based on the size of the cloning cylinder, add between 50 to 200 μl of trypsin. Dislodge the cells by gently pipetting up and down without disturbing the cylinder. For each colony, transfer the trypsinized cells to a single well of a 24 well tissue culture plate containing 1 mL of DMEM (10% v/v FBS). Incubate at 37°C in a CO2 incubator for 22–24hrs. DMEM (10% v/v FBS), as opposed to Selection Medium, is used at this stage in order to ensure viability of the newly picked colony.
  • 17|
    On the next day, exchange the DMEM in each well with 1 mL of Selection Medium. Keep exchanging with fresh Selection Medium every 3–4 days until the cells are confluent.
  • 18|
    At confluency, aspirate off the medium and add 100 μL of trypsin. Transfer the resuspended cells to 1 well of a 6 well tissue culture plate containing 2 mL of Selection Medium. Transfer any residual cells left over in the 24 well using this 2 mL of Selection Medium. Once the cells reach confluency, aspirate off the medium and add 200 μL of trypsin. Transfer the resuspended cells to a 10 cm2 plate possessing 10 mL of the Selection Medium.
  • 19|
    Once the cells reach approximately 90% confluency, spilt the cells into two 15 cm2 tissue culture plates. Incubate at 37°C until the plates reach confluency.
  • 20|
    Use one of the 15 cm2 tissue culture plates to make frozen cell stocks as described in Box 1. Use the other 15 cm2 tissue culture plate for assessing membrane protein expression levels (see step 21–34).

    BOX 1

    Making frozen cell stocks ● TIMING ~30 min

    1. Aspirate off the medium, wash the cells with 10 mL of D-PBS, and add 2 mL of trypsin.
    2. Resuspend the trypsinized cells with 10 mL of DMEM, transfer to a 15 mL centrifuge tube, and pellet the cells at low speed for 4–5 min.
    3. Aspirate off the medium and gently resuspend the cells in 3 mL of cell preservation medium, ensuring that there are no large clumps of cells.
    4. Make three frozen cell stocks by transferring 1 mL of resuspended cells to each of three 1.5 mL cryovials. Transfer the vials to Cryo container and store at −80°C.
    5. After 24 hours, transfer the cryovials to a liquid nitrogen storage dewar. Use these frozen stocks to expand highly expressing clones (step 35).
  • 21|
    Aspirate off the medium, wash the cells with 10 mL of D-PBS, and add 25 mL of DMEM, being careful not to dislodge the cells.
  • 22|
    To induce the cells, add doxycycline (final concentration = 2 μg/mL) and sodium butyrate (final concentration = 5 mM), and incubate at 37°C.
  • 23|
    After 24 hours, aspirate off the medium, wash the cells with 10 mL of D-PBS, and add 2 mL of trypsin.
  • 24|
    Resuspend the trypsinized cells thoroughly in 10 mL of DMEM, and pellet the cells at low speed for 4–5 min.
  • 25|
    Aspirate off all of medium and flash freeze the cell pellet in liquid nitrogen. Store the frozen cells at −20°C until at least 12 clones have been produced before proceeding to step 26 for small scale solubilization and western blotting analysis.
    PAUSE POINT Cell pellets can be stored in a −80°C freezer for weeks, or in a liquid N2 storage dewar for years, for future use.

Assessment of membrane protein solubilization and expression levels from clonal cell lines ● TIMING 1 day

  • 26|
    Remove the frozen cell pellets from −20°C and thaw on ice. Measure the weight of the pellet. Add 0.5 mL of 2X Western solubilization buffer (no detergent) per 0.1 g of cells to each tube.
  • 27|
    Resuspend the pellet with a pipette. Aliquot the resuspended cells equally into two glass tubes containing an equal volume of either 40 mM DDM or 400 mM OG, in H20.
  • 28|
    Place the samples in a water bath sonicator and sonicate for 1 min. Incubate the samples on ice for 1 minute. Repeat the sonication and incubation on ice cycle five times.
  • 29|
    Transfer 400 μL of sonicated sample into a 1.5 mL ultracentrifuge tube containing a small magnetic stir bar. Add 0.2 μL of Benzonase and stir at 4°C for 30 minutes at moderate speed.
  • 30|
    Take a 50 μL aliquot as a “before spin” sample.
  • 31|
    Spin the remaining sample at 200,000g for 20 minutes at 4°C using a Type 45 Ti ultracentrifuge rotor. Transfer to a clean 1.5 mL centrifuge tube labeled as “after spin”.
  • 32|
    For each clone, resolve 10 μL of “before spin” and “after spin” sample by reducing SDS-PAGE.
  • 33|
    Transfer to a nitrocellulose membrane, following the manufacturer’s protocols.
  • 34|
    Perform a western blot using either anti-His or anti-FLAG HRP-conjugated antibodies, following the manufacturer’s protocols. The two clones that possess the highest level of solubilized membrane protein (i.e. the strongest band in the “after spin” lane) are saved for further cell culture scale up (step 35). The remaining clones can either be discarded or saved; we typically save frozen stocks of the eight highest expressing clonal cell lines, until we establish that our best clone expresses from a 1 L spinner flask. The levels of membrane protein expression from each clone can be expected to be highly variable (Figure 4). ▲CRITICAL STEP It is important to immunoblot both the “before spin” and “after spin” samples for each clone, given that the “before spin” sample reveals the amount of membrane protein produced, while the “after spin” sample reveals the amount of this that is solubilized. A low amount of solubilized “after spin” signal, relative to “before spin”, can indicate that either the detergent is not sufficient for solubilization (as is often the case with OG), or that the membrane protein is not well folded. In addition, in cases of low solubilization, additional variables such as different detergents, pH and salt concentration should be assessed for their effect on solubilization.
    Figure 4
    Expression levels from stably transfected and clonal HEK293S GnTI- cell lines as determined by western blotting. Each lane corresponds to the “after spin” sample from a separate clonal cell line (clones 1 to 9), visualized via anti-FLAG ...

Expansion of cells from liquid nitrogen frozen stocks ● TIMING 1 week

  • 35|
    Once the best expressed clone has been determined via western blotting, transfer one vial of cells from the liquid nitrogen storage dewar (see Box 1 ) to dry ice. In the tissue culture room, incubate the vial in a 37°C water bath for ~1–2 min, until the cells are almost completely thawed.
  • 36|
    Remove the vial from the 37°C water bath. Thoroughly clean the outside of the vial with 70% v/v alcohol prior to opening it in the biosafety cabinet.
  • 37|
    Add 0.5–1 mL of DMEM to the vial, resuspend gently and add the resuspended cells with 10 mL of DMEM to a 15 mL centrifuge tube.
  • 38|
    Centrifuge at low speed for 5 min, aspirate off the medium, and resuspend the cells in 10 mL of DMEM medium. Transfer cells to a 10 cm2 tissue culture plate and incubate overnight at 37°C. ▲CRITICAL STEP Inadequate removal of the cell preservation medium prior to seeding can result in a complete loss of cell viability.
  • 39|
    Aspirate off the medium, which should contain many floating, dead cells. Add 10 mL of DMEM and incubate at 37°C until the plate reaches confluency.
  • 40|
    Split cells into two 15 cm2 tissue culture plates (see step 19), and continue splitting until ten confluent 15 cm2 plates are produced.
    ? Troubleshooting

1 L Cell cultures in spinner flasks ● TIMING ~3 days

  • 41|
    Resuspend the cells from ten 15 cm2 tissue culture plates, following the protocol as described in Box 1 with the exception that Suspension Medium is substituted for DMEM. This is typically performed five plates at a time.
  • 42|
    Transfer the cells to a 1L spinner flask containing 500 mL of Suspension Medium.
  • 43|
    Add the remaining Suspension Medium (~400 mL) to the spinner flask, and ensure that the lids of the two spouts of the flask are tightly closed prior to removal of the spinner flask from the biosafety cabinet.
  • 44|
    Place the flask on top of a magnetic stir plate in the 37°C incubator and stir at ~65 RPM. To ensure gas exchange between the incubator and the flask, loosen both caps of the spinner flask, ensuring that enough thread is still held to prevent the caps from being removed from the flask via a straight pull. ▲CRITICAL STEP Ensure that 2 mL of resuspended cells (from a total volume of 12 mL) from one 15 cm2 is used to seed a new 15 cm2 tissue culture plate. This “maintenance plate” can be used in the future to seed further 1 L spinner flasks.
  • 45|
    The next day, measure the cell density, which should be ~0.5 × 106 cells/mL. Continue to incubate at 37°C at ~65 RPM. ▲CRITICAL STEP To avoid contamination, ensure that the lids of the spinner flask are closed prior to removal of the flask from the incubator.
    ? Troubleshooting
  • 46|
    The next day, again measure the cell density, which should now be ~ 0.7–1.0 × 106 cells/mL. At this point, proceed to either generate 2 × 1 L spinner flasks (step 47) or feed and induce the cell culture (step 61), depending on the final scale of cell culture that is desired.

2 L Cell cultures in spinner flasks ● TIMING ~3 days

  • 47|
    Warm 1 L of Suspension Medium to 37°C in a water bath. Transfer half of the cells from a 1 L spinner flask (density = ~ 0.7–1.0 × 106 cells/mL) into a second sterilized 1 L spinner flask. Add 500 mL of Suspension Medium to each spinner flask. Ensure that the lids of both flasks are partially loosened (see step 44) after placing the flasks in the incubator. Incubate overnight at 37°C at ~65 RPM.
  • 48|
    Measure the cell density, which should be ~0.7–1.0 × 106 cells/ml. At this point, proceed to either inoculate a 10 L WAVE bioreactor (step 49), or to feed and induce the cell cultures (step 61). Optionally, additional 1 L or 3 L spinner flasks can be generated and maintained in an incubator (see Figure 5a).
    Figure 5
    Medium and large scale HEK293S GnTI- cell cultures. As shown in panel A, up to 7 × 1 L spinner flasks can be maintained in a single CO2 incubator. Alternatively, up to 3 × 3 L spinner flasks can maintained (not shown). In panel B, 2 × ...

10 L Cell cultures using WAVE cellbag bioreactors ● TIMING ~3 days

  • 49|
    Warm up 7 L of Suspension Medium in a 37°C incubator.
  • 50|
    In the biosafety cabinet, remove the outer packaging from a 10 L cellbag. Clamp the inlet air and exhaust vent filters. Refer to Figure 5b for cellbag and WAVE bioreactor features.
  • 51|
    Pour all 7 L of Suspension Medium into the cellbag. Wipe the opening of the cellbag with sterile 70% v/v ethanol wipes prior to closing the bag.
  • 52|
    Secure the cellbag to the holder tray of the rocking unit, following the manufacturer’s protocols. Connect the air line (mixed with 5% CO2) from the MIX OUT port of the pump to the inlet air filter on the cellbag and unclamp the inlet air filter.
  • 53|
    Attach the filter heater to the exhaust vent filter to reduce condensation and plug it into the FILTER HEATER port on the back panel of the base of rocking unit. ▲CRITICAL STEP Condensation will nevertheless build up on the exhaust vent filter over time. To prevent blockage of the vent, the vent filter should be periodically flicked to remove excess condensation.
  • 54|
    Set the airflow on the pump to 0.25 LPM in order to inflate the cellbag. Set the temperature to 37°C.
  • 55|
    Turn on the rocking unit, and set the rocking speed to 15 RPM. Verify that the rocking unit is functional.
  • 56|
    After ~15–30 min, verify that the cellbag bioreactor is fully inflated (i.e. taut with no creases observed) and secured to the tray and rocking unit. Unclamp the exhaust vent filter and verify that air is being released through pressure control valve by pressing gently on the cellbag.
  • 57|
    After ~1.5 hours, clamp the inlet air and exhaust vent filters, remove the filter heater, turn off the air pump and remove the air line from the inlet air filter. Remove the cellbag from the rocking unit and transfer it to the biosafety cabinet.
  • 58|
    Inoculate the cellbag by slowly pouring the entire contents of the two 1 L spinner flasks (see step 47) into the cellbag. Wipe the opening of the cellbag with sterile 70% v/v ethanol wipes.
  • 59|
    Repeat steps 52 to 56 to inflate the cellbag. Once inflated, set the rocking speed to 22 RPM, and rock overnight.
  • 60|
    To measure the cell density and viability, first stop the rocker and then sterilize the sample port with 70% v/v alcohol wipes. Attach a 5 mL luer syringe to the sterilized sample port, unclamp the sample port tubing, and withdraw ~3–5 mL of cells. Re-clamp the sample port tubing, remove the syringe and sterilize the sample port with 70% v/v alcohol wipes. When the cell density reaches ~1.0 × 106 cells/mL (typically 2–3 days), proceed to feed and induce the cellbag (see step 61). ▲CRITICAL STEP Removing the syringe prior to re-clamping the sample port tubing can result in contamination of the cellbag.

Induction and harvesting of cell cultures ● TIMING ~3 days

  • 61|
    Feed the cells with 20% w/v glucose (100 mL per 10 L wavebag, 10 mL per 1 L spinner flask) and 10% w/v Primatone RL/UF (300 mL per 10 L wavebag, 30 mL per 1 L spinner flask). Incubate for 24 hours at 37°C.
    ? Troubleshooting
  • 62|
    Induce the cells (typical cell density of ~1× 106) with doxycycline (final concentration = 2 μg/ml) and sodium butyrate (final concentration = 5 mM). Incubate for 24–36 hours at 37°C. ▲CRITICAL STEP For RhCG, an incubation period of 36 hours post induction resulted in the highest amount of protein expression. For novel membrane protein targets, the optimal incubation period should be empirically determined.
  • 63|
    Take an aliquot of cells and measure the cell density and viability. Transfer the entire contents of the spinner flask to 1 L and 250 mL centrifuge bottles. Pellet the cells by spinning at 5000g for 10 minutes. Decant the supernatant, wash the pellets with 50 mL of D-PBS and pellet again at 5000 g for 10 minutes. Decant the supernatant and keep the pellet on ice until ready to solubilize.
    ? Troubleshooting

Whole cell membrane solubilization ● TIMING 2 hrs

  • 64|
    Weigh cell pellet. Resuspend the cells with 0.5 mL of lysis buffer per 0.1 g of cells. Add an equal volume of lysis buffer containing 40X critical micelle concentration (CMC) of appropriate detergent, as determined in step 34. Transfer the sample to a Dounce homogenizer and semi-solubilize the sample with 30 strokes. Perform the Dounce homogenization in multiple steps, if the total volume is too large for a single homogenizer. ▲CRITICAL STEP While RhCG was well solubilized in this manner using OG, we have found that other human membrane proteins are very poorly solubilized in OG, and only well solubilized in DDM. Therefore, the optimal [detergent], pH, and [NaCl], as determined in step 34 should be used for solubilization.
  • 65|
    Stir at moderate speed for 1 hour at 4°C. Optionally, aliquot 30 μl as a “before spin” sample for western blotting analysis (see step 34).
  • 66|
    Transfer the entire contents of the beaker to an ultracentrifuge tube, and spin the cells at 75,000g in a Type 45 Ti ultracentrifuge rotor for 45 minutes at 4°C.
  • 67|
    Filter the supernatant (possessing solubilized membrane proteins) through a 0.45 μM filter. Optionally, aliquot 30 μl as an “after spin” sample for western blotting analysis (see step 34).

FLAG affinity and size exclusion chromatography ● TIMING ~1 day

  • 68|
    For every 2 L of suspension culture, wash 1 mL of FLAG resin (i.e. 2 mL of slurry) with SEC buffer (no DTT) following the manufacturer’s protocols.
  • 69|
    Add supernatant from step 67 to the washed FLAG resin. Transfer mixture to 50 mL centrifuge tubes, and gently mix on a Nutator for 2 hours at 4°C.
  • 70|
    Load the entire contents onto a glass Econo-column at 4°C. Collect the flowthrough containing the unbound material.
  • 71|
    Wash the FLAG resin with 10–20 column volumes of SEC buffer (no DTT). Collect washes.
  • 72|
    Elute the protein from the FLAG resin by adding 1 mL of 100 μg/mL FLAG peptide in SEC buffer (no DTT). Repeat five times, eluting the sample in a separate 1.5 mL centrifuge each time. Add DTT to a final concentration of 2 mM to each elution fraction.
  • 73|
    Resolve flowthrough, wash, and elution 1–5 samples by reducing SDS-PAGE. Stain the gel with Coomassie Blue, following standard protocols. Pool elution fractions that possess the overexpressed membrane protein.
    ? Troubleshooting
  • 74|
    Optionally remove the fusion tag(s) by adding the appropriate proteases, following the manufacturer’s recommend concentrations. In the case of RhCG, 10 μL of 3C and 10 μL of thrombin were initially used to test for enzymatic removal of the FLAG and His tags, respectively. Incubate the reaction overnight on a Nutator at 4°C.
  • 75|
    Regenerate and store the FLAG resin for future use following the manufacturer’s protocols.
  • 76|
    Equilibrate a Superdex 10/300 GL size exclusion chromatography (SEC) column with 2 column volumes of SEC buffer at 0.1 ml/min.
  • 77|
    Concentrate the appropriate elutions (from step 73) to a final volume of ~200–400 μL using an ultra centrifugal filtration device with a 50 k cutoff at 4°C. Inject the entire volume onto the equilibrated Superdex 10/300 GL size exclusion column. ▲CRITICAL STEP It is important, if possible, to minimize the number of injections on the size-exclusion column. A large number of dilute injections (i.e. as performed with an autosampler) will result in a larger peak fraction volume. Subsequent concentration of such a large volume of membrane protein can increase the amount of detergent in the concentrated sample, in particular in the case of DDM, which can impede crystallization. In the case of RhCG, however, free detergent micelles were easily separated from RhCG by SEC and did not concentrate readily using the 50 k cutoff filtration device.
  • 78|
    Pool fractions of the desired peak, concentrate to ~5 mg/mL using a centrifugal filtration device with a 50K or 100K cutoff at 4°C, and proceed to crystallization trials using commercial membrane protein screens. Yields of human membrane proteins from HEK293S cells are typically modest, therefore crystallization screening using nanoliter liquid handlers (such as a mosquito®) are of critical importance. We have utilized the mosquito® to screen a variety of detergent solubilized membrane proteins, and have found that the presence of the detergent typically has no adverse impact on liquid handling.

? TROUBLESHOOTING

Troubleshooting advice can be found in Table 1.

TABLE 1
Troubleshooting.

TIMING

The entire protocol, starting from transfection (step 1) to size-exclusion chromatography purification (step 78) takes approximately 8–10 weeks to complete. Once a stable cell line is generated and characterized, however, 1–2 L spinner flask cultures can be generated in ~2 weeks time, while 10 L cellbag cell cultures can be generated in ~3 weeks time. Multiple spinner flask and/or cellbag cell cultures can be grown in parallel, given adequate access to the appropriate equipment (e.g. incubators, WAVE bioreactors, etc.). The timing for each stage of the Procedure is summarized below and in Figure 2.

  • Step 1: Cell seeding ● TIMING ~15 min
  • Steps 2–9: Transfection ● TIMING ~5–7 hr: 45 min Transfection + 4–6 hr incubation + 15 min media exchange
  • Step 10: Expansion ● TIMING ~2 hrs
  • Step 11: Geneticin selection ● TIMING ~15 min
  • Step 12: Generation of stably transfected cell colonies ● TIMING 2–3 weeks
  • Steps 13–25: Generation of clonal cell lines ● TIMING 2–3 weeks
  • Steps 26–34: Assessment of membrane protein solubilization and expression levels from clonal cell lines ● TIMING 1 day
  • Steps 35–40: Expansion of cells from liquid nitrogen frozen stocks ● TIMING 1 week
  • Steps 41–46: 1 L cell cultures in spinner flasks ● TIMING ~3 days
  • Steps 47–48: 1 L cell cultures in spinner flasks ● TIMING ~3 days
  • Steps 49–60: 10 L cell cultures using WAVE cellbag bioreactors ● TIMING ~3 days
  • Steps 61–63: Induction and harvesting of cell cultures ● TIMING ~3 days
  • Steps 64–67: Whole cell membrane solubilization ● TIMING ~3 days
  • Steps 68–78: FLAG affinity and size exclusion chromatography ● TIMING ~1 day

ANTICIPATED RESULTS

We have overexpressed human RhCG, in addition to other human membrane proteins, in HEK293S GnTI- cells using the Procedure described here. For each membrane protein that was expressed, the progression through the protocol was very similar (Figure 2). Approximately 2–3 weeks post drug selection, single foci of cells are typically observed for at least one plating dilution (Figure 3), and clonal cell lines can be generated 2–3 weeks after this. The number of clonal cell lines generated can vary, however, as occasionally only <10–12 individual colonies in total are observed. The amount of membrane protein produced from each clone will likely vary significantly, necessitating the use of western blotting to assess expression levels (Figure 4). While human RhCG was well solubilized using OG, in our experience DDM is a preferred detergent given its greater ability to solubilize membrane proteins from HEK293S cells. For those human membrane proteins that cannot be successfully solubilized with DDM (see Table 1), solubilization using harsher detergents such as FC-14 can be attempted, however, caution should be exercised in these cases given the potential for solubilizing an inactive form of the membrane protein31.

A clonal cell line can typically be utilized indefinitely; human RhCG was expressed from a single, clonal cell line over the course of ~ 1 year with no appreciable loss of protein expression. In addition, cell lines can be frozen at any point, and thawed at a later time, again with no appreciable loss of protein expression. In rare cases, membrane protein expression levels from clonal cell lines have been found to decrease upon continued passage; in these cases care must be exercised to thaw fresh cells, and create new frozen stocks, prior to each cell culture scale up.

The use of both spinner flasks and cellbag WAVE bioreactors (Figure 5) for cell culture scale up is recommended, given that their respective scales complement one another. It is to be expected that cellbag cultures should reach higher density, given its more optimal oxygen transfer; however we did not observe this for the RhCG HEK293S cells. Rarely, for certain clonal cell lines, has viability been observed to decrease significantly following induction with doxycycline (see Table 1). In these cases, the cells should be harvested earlier than normal (see Step 63), which will ultimately result in less membrane protein purified per liter of cell culture medium. The optimal conditions for membrane protein stability (i.e. pH, [salt], [glycerol], etc.) can typically be determined using protein purified from a 1 L spinner flask. For human membrane proteins expressed in HEK293S cells, FLAG affinity purification typically results in higher yields and better purity compared with IMAC purification. Final yields of purified membrane proteins from stably transfected, and clonal HEK293S cells are typically up to ~0.5 mg of protein per L of medium.

Supplementary Material

Full Sequence of pACMV-tetO

Acknowledgments

This work was supported by NIH/NIGMS grants P50 GM73210, U54 GM094625 and R37 GM24485.

Footnotes

AUTHOR CONTRIBUTIONS

S.C., F.G., and R.M.S. designed the experiments. S.C. and F.G. performed the experiments. S.C., J.E.P., F.G., and R.M.S. analyzed the data. V.S. and R.M.S. supervised personnel. S.C., J.E.P., and R.M.S. wrote the paper.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

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