This work was financially supported by the grants K22AI099165 (AWB), P41GM103390 (KWM) and P01GM107012 (KWM) from the National Institutes of Health, and by funds from the Roy J. Carver Department of Biochemistry, Biophysics &#38; Molecular Biology at Iowa State University. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

This protocol is sufficient for expression using either HEK293F or HEK293S cells.&#160;
1. Cell Establishment&#160;
<ol>
	<li>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.
	<ol>
		<li>Operate the incubator shaker at 135 rpm, 80% humidity and at 8.0% CO2 and 37 &#176;C. Turn &#34;on&#34; 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 &#176;C for 1 hr.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Transfer one vial of cells containing HEK293F cells, frozen at -80 &#176;C, onto ice and move to the culture room. Note: HEK293F cells are supplied at a density of 1 x 107 cells/ml.</li>
		<li>Warm the vial gently in water-bath at 37 &#176;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Cell Maintenance: Check Cell Density, Viability and Cell Passages
	<ol>
		<li>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 &#62;80%.</li>
		<li>Sterilize the culture flask with the 70% ethanol in water solution and move it into the biosafety cabinet.</li>
		<li>Using a 1 ml pipette and pipettor, slowly withdraw 100 &#956;l 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.</li>
		<li>Mix 7.5 &#956;l of a Trypan Blue Solution with 7.5 &#956;l of cells. Mix vigorously with 7.5 &#956;l of the cell/Trypan blue mixture and transfer it to the counting slide.</li>
		<li>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</li>
		<li>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.</li>
		<li>Repeat steps 1.1.2 and 1.1.3 of the &#34;1.1 Culture Inoculation&#34; protocol above to prepare materials required for fresh culture medium.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Prepare Materials for Transfection
<ol>
	<li>Preparing HEK293 Cells (Day 1)
	<ol>
		<li>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 (&#62;20 ml) will require a greater volume of culture at this stage.</li>
	</ol>
	</li>
	<li>Preparing Stocks of Materials
	<ol>
		<li>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 &#956;M syringe filter and store at -20 &#176;C for long-term use.</li>
		<li>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:
		<ol>
			<li>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 &#956;g/ml ampicillin. E. coli is grown at 37 &#176;C in a non-humidified, shaking incubator.</li>
			<li>Purify the pGEn2 vector encoding target plasmid DNA according to the manufacturer&#39;s DNA purification protocol.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Prepare stock solution of 220 mM valproic (VPA) acid in water and sterilize by passage through a sterile 0.22 &#956;m filter. Store the solution at -20 &#176;C until further use.</li>
	</ol>
	</li>
</ol>
3. Establishing a Transient Transfection&#160;
<ol>
	<li>Transfection (Day 0)
	<ol>
		<li>Check the cell density by following steps 1.2.2 through 1.2.5 of &#34;1.2. Cell Maintenance&#34; above. Determine the amount of cells for the transfection (2.5-3.0 x 106 live cells/ml with the viability &#62;95%). See a sample calculation in Supplementary Materials.</li>
		<li>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.</li>
		<li>In the meantime, prepare a fresh transfection culture medium following step 1.1.3 according to &#34;1.1. Culture Inoculation&#34; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#956;g/&#956;l. 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.</li>
		<li>Add plasmid DNA (300 &#956;l of 0.5 &#956;g/&#956;l) to the culture using micro-pipette and mix by gentle manual shaking. Add PEI (900 &#956;l of 0.5 &#956;g/&#956;l) 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.</li>
		<li>Clean the biosafety cabinet according to Step 1.1.</li>
	</ol>
	</li>
	<li>Dilution (Day 1) (For a 50 ml transfection volume)
	<ol>
		<li>Incubate the transfected culture for 24 hr.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Expression and Harvest (Days 2-6)
	<ol>
		<li>Incubate the cells for an additional 4-5 days (5-6 days total since transfection).</li>
		<li>Withdraw aliquots of culture medium following step 1.2.2. to 1.2.5. described in &#34;1.2. Cell Maintenance,&#34; 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).</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Protein Purification
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Collect a 10 &#956;l 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.</li>
	<li>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.</li>
	<li>Wash the column with 10 column volumes of buffer A and store for future use.</li>
	<li>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 (&#60;2 ml) in the eluted buffer. Use the centrifugal filters to exchange the buffer with buffer A.</li>
	<li>Store purified protein at 4 &#176;C until next use. 
	Note: Protein expressed in the supernatant can be purified by affinity columns selected on the basis of protein&#39;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.</li>
</ol>
5. Analysis of Protein by SDS-PAGE
<ol>
	<li>Dilute the aliquots (7.5 &#956;l) of each sample with an equal volume of 2x loading buffer (0.125 M Tris, pH 8.0, 4% SDS, 10% &#946;-mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue).</li>
	<li>Boil the mixture at 90 &#176;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 &#956;l of marker protein and 14 &#956;l of samples for analyses using a 20 &#956;l pipette with a disposable tip.</li>
	<li>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.</li>
	<li>Stain the gel with staining solution (40% ethanol, 10% acetic acid and 0.1% coomassie brilliant blue) for 2 hr and destain in water.</li>
</ol>
6. Glycans Analysis by Mass Spectrometry
<ol>
	<li>Analyze glycans as described previously44. Briefly, 75 &#956;g 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).</li>
</ol>
7. Protocol Adjustments for Special Scenarios
<ol>
	<li>Labeling Proteins with 15N-labeled Amino Acids (for a 50 ml transfection volume)
	<ol>
		<li>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 &#176;C for 15 min. Allow the solution to cool approximately to 50-60 &#176;C.</li>
		<li>For a 50 ml transfection volume, follow step 3.1.3 according to &#34;3.1 Establishing a transient transfection&#34;. See the Supplementary Materials section for an example with appropriate volumes.</li>
		<li>For transfection, follow steps of &#34;3. Establishing a transient transfection&#34; using fresh culture medium substituted with labeled amino acid residues as prepared above.</li>
		<li>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.</li>
		<li>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</li>
	</ol>
	</li>
	<li>Supplement the Transfection with a Small Molecule
	<ol>
		<li>Prepare 100 mM stock solution of 2-deoxy-2-fluoro-l-fucose using autoclaved water and sterilize this solution using a 0.22 &#956;M filter.</li>
		<li>For a 50 ml culture, add 125 &#956;l (at 250 &#956;M) 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 &#62;90% reduction in fucosylation by adding the fucose analog at a concentration of 250 &#956;M in the transfection and dilution media.</li>
	</ol>
	</li>
</ol>

All authors declare no competing financial interests in this manuscript.

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 &#215;...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/53568">High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension</a>. The Authors section was updated. from:
Ganesh P. Subedi1 
Roy W. Johnson2 
Heather A. Moniz2 
Kelley W. Moremen2 
Adam Barb1 
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 
2Complex Carbohydrate Research Center, University of Georgia
to
Ganesh P. Subedi1 
Roy W. Johnson2 
Heather A. Moniz2 
Kelley W. Moremen2 
Adam W. Barb1 
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 
2Complex Carbohydrate Research Center, University of Georgia

An erratum was issued for:&#160;<a href="https://www.jove.com/t/53568">High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension</a>. The Authors section was updated.

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

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 described1-4. 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 &#62;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 synthetic8-10, to chemoenzymatic11-14 or recovery from engineered recombinant systems15-20, 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 production4,21,22. 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 phosphates24,25, cationic polymers (DEAE-dextran, polybrene, polylysine, polyethylimine (PEI)) or positively charged cationic liposomes26-29. 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 cytoplasm26,30.
Until recently, transient transfection in suspension culture was carried by prior DNA/PEI complex formation followed by addition to the cell culture29. However, W&#252;rm and coworkers reported a highly efficient protocol optimized for recombinant protein production in HEK293 cells that formed a DNA/PEI complex in situ31,32. 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 processing36,37. 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 processing38-41.
The protocol reported here uses the pGEn2 vector as shown in Figure 142,43, 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.

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

high yield expression of recombinant human proteins with the transient transfection of hek293 cells in suspension

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 Fc&#947;RIIIa and the rat &#945;2-6 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.

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 ...

Watch this Scientific Journal Video about High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension at JoVE.com

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

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.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

high-yield-expression-recombinant-human-proteins-with-transient

Research

JoVE Journal

Biology

30.0K Views.  Iowa State University. 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.

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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Genetics

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Biochemistry

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

Neuroscience

Crude Vector Production and Efficient DNA Delivery in Cell Cultures

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

Author Spotlight: Unveiling the Potential of Unpurified Recombinant AAVs in Cell Culture Research 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

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

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