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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Medium A: FreeStyle Expression Medium&#160;</td>
			<td>Life Technologies</td>
			<td>12338-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium B: ExCell 293 Serum-Free Medium&#160;</td>
			<td>SIGMA</td>
			<td>14571C&#160;</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&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides&#160;</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter&#160;</td>
			<td>Bio-Rad&#160;</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 &#956;M syringe filter and store at -20 &#176;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 &#956;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&#160;</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&#160; in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Corning&#63720; 250 mL Centrifuge Tube&#160;</td>
			<td>Corning Incorporated/Life Sciences</td>
			<td>430776</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific&#160;</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&#174; Ultra centrifugal filters&#160;</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&#160;</td>
			<td>Fisher Scietific</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>coomassie brilliant blue&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0406</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOFMS Voyager-DE PRO&#160;</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15N labeled L-Tyrosine</td>
			<td>ALDRICH</td>
			<td>332151</td>
			<td></td>
		</tr>
		<tr>
			<td>15N 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>13C6-Glucose</td>
			<td>ALDRICH</td>
			<td>389374</td>
			<td></td>
		</tr>
		<tr>
			<td>2-deoxy-2-fluoro-l-fucose&#160;</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 ...

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Research

JoVE Journal

Biology

29.4K 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

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

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) ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

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. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...