High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Summary

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

Abstract

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Introduction

Producing high yields of appropriately folded and post-translationally modified human proteins for detailed analysis of structure and function remains a significant challenge. A large number of expression systems are available that produce recombinant proteins with native-like function and behavior. Bacterial expression systems, predominantly Escherichia coli strains, represent the most accessible and commonly used tools in the research arena, due to the simplicity of these expression systems, though yeast, plant, insect and mammalian systems are also described^1-4. However, the majority of these systems are incapable of appropriate post-translational modification of the target proteins. A fundamental interest of the Barb and Moremen laboratories is producing eukaryotic proteins with appropriate glycosylation. Many human proteins require appropriate glycosylation for proper function (see⁵).

The eukaryotic glycosylation machinery is extensive and capable of making a diverse range of modifications, including both asparagine(N)- and serine/threonine(O)-linked complex glycans⁶. It is estimated that >50% of human proteins are N-glycosylated⁷. Glycans are essential components of many proteins including therapeutic monoclonal antibodies, erythropoietin, and blood clotting factors like factor IX, to name a few. Though multiple methods exist to prepare appropriately N-glycosylated proteins and range from purely synthetic^8-10, to chemoenzymatic^11-14 or recovery from engineered recombinant systems^15-20, not surprisingly, human expression systems have thus far proven to be the most robust methods for generating human proteins.

Many therapeutic human glycoproteins are produced in recombinant systems using mammalian cells. Systems of note are the Chinese Hamster Ovary (CHO), mouse myeloma (NS0), Baby Hamster Kidney (BHK), Human Embryonic Kidney (HEK-293) and human retinal cell lines that are employed in adhesion or suspension culture for protein production^4,21,22. However, mammalian protein expression systems have required the generation of stable cell lines, expensive growth media and substrate assisted transfection procedures²³.

Mammalian cell transfection is achieved with the aid of numerous agents including calcium phosphates^24,25, cationic polymers (DEAE-dextran, polybrene, polylysine, polyethylimine (PEI)) or positively charged cationic liposomes^26-29. PEI is a polycationic, charged, linear or branched polymer (25 kDa)²⁶ that forms a stable complex with DNA and is endocytosed. Upon acidification of the endosome, PEI is thought to swell, leading to the rupture of endosomes and release of the DNA into the cytoplasm^26,30.

Until recently, transient transfection in suspension culture was carried by prior DNA/PEI complex formation followed by addition to the cell culture²⁹. However, Würm and coworkers reported a highly efficient protocol optimized for recombinant protein production in HEK293 cells that formed a DNA/PEI complex in situ^31,32. This avoided preparation, sterilization of the complex, and buffer exchange into a culture medium. Further optimization by including expression-enhancing plasmids led to significant yield increases³³. Herein is a method that builds upon these advances and is broadly applicable. Expression conditions may also be altered to impact the N-glycan composition.

The HEK293S cell line, with a gene deletion that halts N-glycan processing at an intermediate stage, leads to the expression of proteins with uniform N-glycans consisting of 2 N-acetylglucosamine residues plus five mannose residues (Man₅GlcNAc₂)^34,35. These cells lack the N-acetylglucosaminyl transferase I (GntI) gene which is required for downstream N-glycan processing^36,37. The use of glycosyltransferase inhibitors including kifunensine, sialic acid analogs and the fucose analog and 2-deoxy-2-fluoro-fucose has similar effects and limits N-glycan processing^38-41.

The protocol reported here uses the pGEn2 vector as shown in Figure 1^42,43, PEI assisted transient transfection into mammalian cells lines (HEK293F or HEK293S cells), and the recovery of high yields of appropriately glycosylated protein. This system is robust and can accommodate various factors including isotope labeling and glycan engineering for the production of large titers of recombinant proteins.

Protocol

This protocol is sufficient for expression using either HEK293F or HEK293S cells. 

1. Cell Establishment 

1. Culture Inoculation
   Note: All culture manipulation procedures must be carried in a BSL-2 facility and each item brought into the biosafety cabinet must be sterilized by spraying with a 70% ethanol in water solution.
   1. Operate the incubator shaker at 135 rpm, 80% humidity and at 8.0% CO₂ and 37 °C. Turn "on" the UV lamp of the biosafety cabinet at least 1 h prior to working. Prewarm the sealed Medium A and Medium B bottles in the water-bath at 37 °C for 1 hr.
   2. Sterilize 125 ml Erlenmeyer growth flasks with a vented cap, pipettes, pipettors, and prewarmed Media bottles by spraying with the 70% ethanol in water solution. Place these items in the biosafety cabinet. Note: Sterilize only the outer plastic covering the 125 ml Erlenmeyer culture flask, and then remove only when it is inside the biosafety cabinet.
   3. For a 30 ml culture, withdraw 26 ml of Medium A and 3 ml of Medium B (10% of the final culture) and transfer into the 125 ml Erlenmeyer culture flask and gently mix by shaking (hereafter termed as fresh culture medium).
   4. Transfer one vial of cells containing HEK293F cells, frozen at -80 °C, onto ice and move to the culture room. Note: HEK293F cells are supplied at a density of 1 x 10⁷ cells/ml.
   5. Warm the vial gently in water-bath at 37 °C to partially thaw the cells (it takes approximately one min and cells should not be completely thawed). Sterilize the outside of the vial with the 70% ethanol in water solution and move it into the biosafety cabinet.
   6. Using 1 ml pipette, withdraw the cell suspension (approximately 0.7 ml) and transfer into the 125 ml Erlenmeyer flask containing 29 ml of fresh culture medium. Close the cover of the culture flask and move it into the incubated shaker.
2. Cell Maintenance: Check Cell Density, Viability and Cell Passages
   1. Check the cells density after 24 hr of thawing of the culture by following the protocol below. Note: Cells are grown for 24 hr after thawing to ensure essential cells growth and viability of >80%.
   2. Sterilize the culture flask with the 70% ethanol in water solution and move it into the biosafety cabinet.
   3. Using a 1 ml pipette and pipettor, slowly withdraw 100 μl of suspension culture and transfer into a sterile 0.5 ml eppendorf tube. Close and transfer the culture flask back into the shaker as soon as possible.
   4. Mix 7.5 μl of a Trypan Blue Solution with 7.5 μl of cells. Mix vigorously with 7.5 μl of the cell/Trypan blue mixture and transfer it to the counting slide.
   5. Turn on the automatic cell counter and place the counting slide into the loading chamber. The auto reader is activated and cells are counted automatically in the units of number of cells per ml and percent viability
   6. Determine the volume of the donor culture required for the transfer to a fresh culture medium at the cells density of 0.3 x 10⁶ live cells/ml.
      Note: See a sample calculation in Supplementary Materials.
   7. Repeat steps 1.1.2 and 1.1.3 of the "1.1 Culture Inoculation" protocol above to prepare materials required for fresh culture medium.
   8. Transfer Medium A (23 ml) and Medium B (3 ml) to a fresh 125 ml culture flask. Remove the stock bottles of Medium A and Medium B from the biosafety cabinet.
      Note: Stocks of media bottles should not be kept in the biosafety cabinet at the same time as the HEK 293F cells. This limits the possibility of contaminating the stock medium bottles.
   9. Remove the HEK293 cell culture from the incubator and spray with the 70% ethanol in water solution. Using a new pipette, withdraw the aliquot of cells (4 ml, this volume was determined according to step 1.2.6) from the culture and transfer it to the flask containing fresh culture medium.
   10. Transfer both the cultures from the biosafety cabinet to the incubator shaker set according to 1.1.1. Grow cells to an approximate density of 2-3 x 10⁶ live cells/ml.
       Note: The approximate doubling time of cells is 32 hr. It will take 3-4 days to reach this density. Incubate the cells for 3-4 passages to have stable growth pattern of cells prior to the first transfection.

2. Prepare Materials for Transfection

1. Preparing HEK293 Cells (Day 1)
   1. Subculture cells to a density of 1 x 10⁶ cells/ml in fresh Medium B 24 hr prior to transfection according to Step 1.2.
      Note: the volume of the transfection culture will be dependent upon the number of cells. Larger transfection volumes (>20 ml) will require a greater volume of culture at this stage.
2. Preparing Stocks of Materials
   1. Prepare stock solution of linear Polyethylenimine (PEI) at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; this may take 30-60 min at RT. Sterilize through 0.22 μM syringe filter and store at -20 °C for long-term use.
   2. Prepare sterile plasmid DNA (according to steps 2.2.2.1-2.2.2.4). The plasmid construction procedure is described elsewhere⁴². A brief procedure for DNA preparation is explained here:
      1. Grow a culture of chemically-competent Escherichia coli cells transformed with the pGEn2 vector in 1 L of LB medium (Tryptone- 10 g/L, yeast extract- 5 g/L and sodium chloride- 10 g/L) supplemented with 100 μg/ml ampicillin. E. coli is grown at 37 °C in a non-humidified, shaking incubator.
      2. Purify the pGEn2 vector encoding target plasmid DNA according to the manufacturer's DNA purification protocol.
      3. To ensure complete sterility of the plasmid DNA, perform the final isopropanol precipitation and resuspension steps of the DNA extraction procedures inside the biosafety cabinet.
      4. Add the volume of isopropanol (specified in the plasmid preparation kit) to the eluted DNA and centrifuge at 10,000 x g for 10 min. Sterilize the outside of the DNA container with 70 % ethanol in water solution and transfer it inside the biosafety cabinet. Discard the isopropanol supernatant using a pipette and air-dry the DNA pellet. Once dry, resuspend the pellet in sterile 10 mM Tris buffer, pH 8.0.
   3. Prepare stock solution of 220 mM valproic (VPA) acid in water and sterilize by passage through a sterile 0.22 μm filter. Store the solution at -20 °C until further use.

3. Establishing a Transient Transfection 

1. Transfection (Day 0)
   1. Check the cell density by following steps 1.2.2 through 1.2.5 of "1.2. Cell Maintenance" above. Determine the amount of cells for the transfection (2.5-3.0 x 10⁶ live cells/ml with the viability >95%). See a sample calculation in Supplementary Materials.
   2. Transfer the volume of suspension culture cells calculated in the previous step (here 67 ml) to a 250 ml centrifuge tube using a sterile serological pipette. Collect cells by centrifugation for 5 min at 100 x g.
   3. In the meantime, prepare a fresh transfection culture medium following step 1.1.3 according to "1.1. Culture Inoculation" above. See the Supplementary Materials for a sample calculation. Also, transfer 5 ml of fresh culture medium to a sterile 15 ml tube and set aside.
   4. Transfer tubes containing the pelleted cells into the biosafety cabinet after sterilizing the outside of the tubes with the 70% ethanol in water solution and use a sterile pipette to decant the supernatant consisting of spent culture medium.
   5. Vigorously resuspend the pelleted cells by pipetting up and down using 10 ml of fresh culture medium and transfer to the flask with fresh transfection culture medium (prepared in step 3.1.3 above). Screw the cap on the vented cell culture flask and move the flask to the incubator (as set in 1.1.1) for 15 min-1 hr with shaking.
   6. During this time, dilute the plasmid DNA and PEI stocks using fresh culture medium (using the 5 ml volume withdrawn from step 3.1.3 above) to a final concentration of 0.5 μg/μl. See sample calculations in Supplementary Materials to determine the amount of DNA and PEI. Transfer the culture flask with the dispersed cells into the biosafety cabinet.
   7. Add plasmid DNA (300 μl of 0.5 μg/μl) to the culture using micro-pipette and mix by gentle manual shaking. Add PEI (900 μl of 0.5 μg/μl) to the culture, mix by gently shaking. Add 3.8 ml of fresh culture medium, from step 3.1.3, above and transfer flask to the incubator shaker for 24 hr.
   8. Clean the biosafety cabinet according to Step 1.1.
2. Dilution (Day 1) (For a 50 ml transfection volume)
   1. Incubate the transfected culture for 24 hr.
   2. Withdraw 1 ml of prewarmed 220 mM Valproic Acid (VPA; prepared according to step 2.2.3.) using a sterile 1 ml serological pipette and transfer it to a sterile 50 ml tube.
   3. Withdraw 5.0 ml of prewarmed Medium B and 44 ml of prewarmed Medium A and add this to the VPA solution using a sterile serological pipette to prepare 50 ml of fresh culture medium with a final concentration of 4.4 mM of VPA.
   4. Move the transfected culture into the biosafety cabinet after sterilizing the exterior of the flask with a 70% ethanol in water solution, add the prepared dilution medium and transfer the flask back to the incubated shaker.
3. Expression and Harvest (Days 2-6)
   1. Incubate the cells for an additional 4-5 days (5-6 days total since transfection).
   2. Withdraw aliquots of culture medium following step 1.2.2. to 1.2.5. described in "1.2. Cell Maintenance," above using a sterile 1 ml serological pipette, to monitor cells viability and save aliquots to analyze protein expression at each day by SDS-PAGE (see Section 5, below).
   3. Harvest cells after 5-6 days by centrifuging the cells plus medium at 1,000 g for 5 min. Additionally, harvest the supernatant if the cell viability falls below 50% (see steps 1.2.2-1.2.5).
   4. Pour off the supernatant that will contain secreted protein. Add 5 ml of a 10% bleach to the HEK cell pellet and discard as a biohazard.

4. Protein Purification

1. Prepare a column with 5 ml of Protein-A sepharose. Equilibrate the column with 5 column volumes of buffer A (20 mM of 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.4, 100 mM NaCl).
2. Centrifuge the collected supernatant with expressed protein at high speed (14,000 g for 10 min) to remove any remaining cell debris. Collect by decanting into a fresh tube. Discard the pellet as a biohazard.
3. Collect a 10 μl aliquot of the supernatant and additionally collect a small sample at each protein purification step to analyze the sample by SDS-PAGE (described in Section 5). Load the clarified supernatant and collect the flow through.
4. Wash the column with 3-column volumes of buffer A and elute the protein with 5-column volumes of 100 mM glycine, pH 3.0. Collect the eluate in tubes containing one-half volume of 1 M TRIS buffer, pH of 8.0 to rapidly neutralize the pH.
5. Wash the column with 10 column volumes of buffer A and store for future use.
6. Buffer exchange the eluted protein with at least a 10-fold excess of buffer A using 10 kDa molecular weight cut-off centrifugal filters. Note: Do not concentrate the eluted sample to a very low volume (<2 ml) in the eluted buffer. Use the centrifugal filters to exchange the buffer with buffer A.
7. Store purified protein at 4 °C until next use.
   Note: Protein expressed in the supernatant can be purified by affinity columns selected on the basis of protein's properties or the use of affinity tags. For a typical purification procedure, Protein A column can be used for IgG or Fc fragments or nickel column can be used for proteins expressed with a poly-histidine tag. Here, is a description of the purification steps for IgG1-Fc using a Protein A column.

5. Analysis of Protein by SDS-PAGE

1. Dilute the aliquots (7.5 μl) of each sample with an equal volume of 2x loading buffer (0.125 M Tris, pH 8.0, 4% SDS, 10% β-mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue).
2. Boil the mixture at 90 °C for 5 min using a heat block or water bath. Centrifuge the samples at 10,000 g for 1 min. Load the prepared gel with 5 μl of marker protein and 14 μl of samples for analyses using a 20 μl pipette with a disposable tip.
3. Run one gel at 25 milliamps for 60 min. Once the electrophoresis step is complete, transfer the gel to a container with 1 L of water and microwave for 5 min.
4. Stain the gel with staining solution (40% ethanol, 10% acetic acid and 0.1% coomassie brilliant blue) for 2 hr and destain in water.

6. Glycans Analysis by Mass Spectrometry

1. Analyze glycans as described previously⁴⁴. Briefly, 75 μg of IgG1-Fc was trypsinized, then treated by PNGaseF for the release of glycans⁴⁵. Released glycans were permethylated and analyzed by using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS).

7. Protocol Adjustments for Special Scenarios

1. Labeling Proteins with ¹⁵N-labeled Amino Acids (for a 50 ml transfection volume)
   1. Dispense 5 mg of each amino acid into a single sterile glass vial. Resuspend with 4 ml autoclaved water (note Tyr does not completely solubilize, unlike Lys and Phe). Sterilize the solution by autoclaving at 121 °C for 15 min. Allow the solution to cool approximately to 50-60 °C.
   2. For a 50 ml transfection volume, follow step 3.1.3 according to "3.1 Establishing a transient transfection". See the Supplementary Materials section for an example with appropriate volumes.
   3. For transfection, follow steps of "3. Establishing a transient transfection" using fresh culture medium substituted with labeled amino acid residues as prepared above.
   4. After 24 hr of transfection, on the day of culture dilution, withdraw 5.0 ml of prewarmed Medium B, 40 ml of prewarmed Medium A and a freshly autoclaved 4 ml aliquot of the amino acid mixture (prepared as in step 7.1.1. above) and add 1 ml prewarmed stock solution of VPA solution to prepare 50 ml of fresh dilution culture medium with a final concentration of 4.4 mM of VPA. Add this medium to the transfection culture using a sterile serological pipette.
   5. Transfer the transfected culture into the biosafety cabinet after sterilizing the exterior of the flask with a 70% ethanol in water solution, add the prepared dilution medium and transfer the flask back to the incubated shaker.
      Note: Medium A is a chemically defined, serum-free medium. Thus, it is possible to prepare a different formulation. Contact the supplier to obtain a custom Medium A preparation that lacks certain amino acids. It is possible to have all amino acids left out, however, we prefer to use medium that lacks only few select residues because a complete dropout medium would require adjustment of osmolarity after supplementation. We chose a Lys-Tyr-Phe dropout medium that can be supplemented and does not require osmolarity adjustment. Furthermore, the Medium A supplier does not freely share the concentrations of medium components; we used 100 mg/L each for Lys, Tyr and Phe with success. Volumes of the transfection and expression medium must be adjusted to account for the added amino acids. Here are the adjustments to the protocol above for isotope labeling
2. Supplement the Transfection with a Small Molecule
   1. Prepare 100 mM stock solution of 2-deoxy-2-fluoro-l-fucose using autoclaved water and sterilize this solution using a 0.22 μM filter.
   2. For a 50 ml culture, add 125 μl (at 250 μM) of the fucose analog solution to the transfection and dilution media. Note: We neglected to perform any further volume adjustment because adding the substituent at this concentration accounts for only 0.25% of the total culture volume .It is possible to modify the expression using small molecule inhibitors of glycan processing. Here we describe the conditions for including 2-deoxy-2-fluoro-l-fucose, an inhibitor of glycan fucosylation. We found >90% reduction in fucosylation by adding the fucose analog at a concentration of 250 μM in the transfection and dilution media.

Results

High-level protein expression and purity

This optimized expression system generated a high yield of glycosylated proteins. A typical pattern is shown in the expression of IgG1-Fc (Figure 1). In this case, Day 0 is the transfection day followed by Day 1 (dilution) and subsequent culture days up to Day 5. Protein expression is analyzed using the soluble expression fraction in the crude medium. A very small amount ...

Discussion

This protocol illustrates protein expression via the transient transfection of HEK293F or S cells. The optimal transfection conditions established in the Barb and Moremen labs employ a critical combination of cell density and reagent concentrations to achieve high efficiency transfection. Critical considerations when implementing this protocol include: maintaining a stable culture prior to transfection (with consistent culture doubling times); transfection of actively growing cells (achieved by diluting cells to 1 ×...

Materials

Name	Company	Catalog Number	Comments
Biosafety cabinet	NuAire, Inc.	CellGard ES NU-S475-400	Class II, Type A2 Biological Safety Cabinet
Incubation shaker	INFORS HT		Multitron Cell
Medium A: FreeStyle Expression Medium	Life Technologies	12338-018
Medium B: ExCell 293 Serum-Free Medium	SIGMA	14571C
125 Erlenmeyer Flask with Vented Cap	Corning Incorporated/Life Sciences	431143
250 Erlenmeyer Flask with Vented Cap	Corning Incorporated/Life Sciences	431144
FreeStyle HEK 293F Cells	Life Technologies	R790-07
1 ml Disposable serological pipette	Fisher Scietific	13-676-10B
10 ml Disposable serological pipette	Fisher Scietific	13-676-10J
25 ml Disposable serological pipette	Fisher Scietific	13-676-10K
Pipettor (Pipet-Aid XP)	Drummond Scientific	161263
Trypan Blue Solution	Thermo Scientific	SV30084.01
Counting slides	Bio-Rad	145-0011
TC20 Automated Cell Counter	Bio-Rad	145-0102
Polyethylenimine (PEI)	Polysciences Inc.	23966	Prepare stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; sterilize through 0.22 μM syringe filter and store at -20 °C.
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	EMD Chemicals Inc.	7365-45-9
25 mm Syringe Filter, 0.22 μM	Fisher Scietific	09-719A
Trisaminomethane (Tris base)	Fisher Scietific	BP152-1
XL1-Blue	Stratagene	222249
Trypton	Fisher Scietific	BP1421-2
Yeast extract	Fluka Analytical	92144
Sodium chloride	BDH chemicals	BDH8014
Plasmid Purification Kit	QIAGEN	12145
Valproic (VPA)	SIGMA	P4543	Prepare stock solution of 220 mM  in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 °C
Corning 250 mL Centrifuge Tube	Corning Incorporated/Life Sciences	430776
Centrifuge	Thermo Scientific	EW-17707-65
Protein A-Sepharose column	SIGMA	P9424
Ni-NTA superflow	QIAGEN	30430
3-(N-morpholino)propanesulfonic acid (MOPS)	Fisher Scietific	BP308-500
Glycine	Fisher Scietific	BP381-500
10 kDa molecular weight cut-off Amicon® Ultra centrifugal filters	Millipore	UFC901096
Sodium dodecyl sulfate	ALDRICH	L3771
Beta-mercaptoethanol	ALDRICH	M6250
Glycerol	SIGMA	G5516
Precision Plus Protein All Blue Standards	Bio-Rad	161-0373
Acetic Acid, Glacial	Fisher Scietific	64-19-7
coomassie brilliant blue	Bio-Rad	161-0406
MALDI-TOFMS Voyager-DE PRO	Applied Biosystems
15N labeled L-Tyrosine	ALDRICH	332151
15N labeled L-Lysine	ALDRICH	592900
Unlabeled L-Phenylalanine	SIGMA-ALDRICH	P2126
13C6-Glucose	ALDRICH	389374
2-deoxy-2-fluoro-l-fucose	SANTA CRUZ Biotechnology	sc-283123