Name: recombinant protein expression for structural biology in hek 293f suspension cells: a novel and accessible approach
Uploaded: 2014-10-16T15:00:00
Duration: 11 min 20 s
Description: Watch this Scientific Journal Video about Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach at JoVE.com

We would like to thank Dr. Xiaowen Yang (PROTEX cloning service) for preparing the expression constructs used for this work and the University of Leicester Core Biotechnology Services. This work was supported by a BBSRC studentship, the Wellcome Trust Programme and Senior Investigator grants WT085408 &#38; WT100237 and BBSRC grant RM31G0224.

NOTE: The protocol is suitable for any scale of expression, therefore volumes and quantities of reagents should be scaled proportionally. A suitable mammalian expression vector must be used for this protocol. Here we used a modified pcDNA 3.1 expression vector that conveniently allows the inclusion or exclusion of an affinity tag depending on the choice of restriction enzymes used in the cloning (Figure 1). The following protocol describes a large scale transfection.
1. Large Scale Culture/Transfection in 1 L Roller Bottles
Grow and maintain HEK293F suspension-adapted cells according to standard protocols. Typically starter cultures of between 30 and 100 ml are grown in 250 ml conical cell culture flasks.
<ol>
	<li>Seed cells at 0.5 x 106 cells/ml into a final volume of 300 ml in each 1 L roller bottle. 
	NOTE: Roller bottles accommodate a minimum of 150 ml to a maximum of 300 ml of suspension culture. For larger scale transfections use multiple bottles.</li>
	<li>Incubate for 24 hr in an orbital shaker incubator at 37 &#176;C, 120 rpm, and 5% CO2 until cells reach a density of 1.0 x 106 cells/ml (cells should divide approximately every 24 hr).</li>
	<li>Pipette a total of 300 &#181;g of filter-sterilized DNA (see supplementary&#160;method)&#160;into 30 ml of PBS and vortex vigorously for 3 sec. 
	NOTE: Use a total of 1 &#181;g of DNA per million transfected cells. If two or more plasmids are to be co-transfected reduce the amount of each so that the ratio of DNA to cells remains constant.&#160;</li>
	<li>Add 1.2 ml of 0.5 mg/ml filter-sterilized PEI to the PBS/DNA solution and vortex vigorously for 3 sec.</li>
	<li>Incubate the mix at RT for 20 min.</li>
	<li>Add the DNA/PEI mix to the cells &#8211; which should be at a density of 1 x 106 cells/ml (step 1.3).</li>
	<li>Following co-transfection, incubate the cells in an orbital shaker incubator for a further 48 hr at 37 &#176;C, 120 rpm, and 5% CO2.</li>
	<li>Harvest intracellular proteins by centrifuging cells at 3,000 x g for 5 min and store the pellet at -80 &#176;C. 
	NOTE: Alternatively, use a standard (non-CO2) shaking incubator, if the atmosphere above the culture is replaced with 5% CO2 and the bottle lid is sealed. The atmosphere will need to be replaced at each passage (normally every 2 days).</li>
</ol>
2. Protein-complex Purification from Whole Cell Extract
This protocol is optimized for the purification of nuclear complexes using flag-tagged proteins.
<ol>
	<li>Defrost cell pellet into ~40 ml of pre-chilled lysis buffer (100 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol, 0.3% Triton X-100, protease inhibitors) per L of culture.</li>
	<li>Re-suspend the pellet by pipetting up and down several times (to avoid foaming, do not vortex).</li>
	<li>Thoroughly re-suspend cells using a glass homogenizer. Sonicate for 3 cycles (15 sec on, 15 sec off). Centrifuge at 30,000 x g for 25 min at 4&#186;C and retain supernatant.</li>
	<li>Equilibrate 1.25 ml of anti-flag packed agarose resin per liter of culture by washing three times with resin equilibration buffer (100 mM potassium acetate, 50 mM Tris pH 7.5).</li>
	<li>Incubate the whole cell extract from step 2.3 with the affinity resin in one or more 50 ml centrifuge tubes and gently rotate the sample for 30-120 min at 4 &#176;C.</li>
	<li>Centrifuge at 3,000 x g for 1 min at 4 &#176;C, discard supernatant.</li>
	<li>Wash the resin with 45 ml of pre-chilled buffer 1 (100 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol, 0.3% Triton X-100). Centrifuge at 3,000 x g for 1 min and discard supernatant.</li>
	<li>Repeat step 2.7 using high salt buffer (300 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol), followed by low salt buffer (50 mM potassium acetate, 50 mM Tris pH 7.5, 5% glycerol) and TEV Cleavage Buffer (50 mM potassium acetate, 50 mM Tris pH 7.5, 0.5 mM TCEP). 
	NOTE: Ensure each wash is brief i.e., sufficient to fully re-suspend the resin and no longer.</li>
	<li>Collect a 10 &#181;l sample of resin and dilute into 1 volume of 2x protein loading buffer for analysis (bound protein control). Do not use reducing agents in order to avoid releasing the antibody from the resin.</li>
	<li>Re-suspend the resin into 8-10 ml of pre-chilled TEV cleavage buffer and transfer to&#160;a 15 ml centrifuge tube.</li>
	<li>Add ~40 &#181;g of TEV protease (from stock at 1 mg/ml) per L of original culture and mix well by pipetting up and down several times.</li>
	<li>Replace tube atmosphere with 100% N2 gas to prevent protein oxidation.</li>
	<li>Gently rotate O/N at 4&#186;C.</li>
	<li>Centrifuge at 3,000 x g for 10 min. Transfer the supernatant into an ultra centrifugal filter with an appropriate molecular weight cut-off and concentrate down to 500 &#181;l.</li>
	<li>Collect a 10 &#181;l sample of the concentrated protein and dilute into 1 volume of 2x protein loading buffer for analysis. (TEV eluate control).</li>
	<li>Collect a 10 &#181;l sample of the resin and dilute into 1 volume of 2x protein loading buffer for analysis. Do not use reducing agents in order to avoid releasing large amounts antibody from the resin. (Post-TEV resin control).</li>
	<li>Equilibrate the size exclusion chromatography column with gel filtration buffer (50 mM potassium acetate, 50 mM Tris pH 7.5, 0.5 mM TCEP).</li>
	<li>Filter the protein through a 0.22 &#181;m filter</li>
	<li>Load the sample into the column and collect the fractions.</li>
	<li>Run samples from steps 2.9, 2.15 and 2.16 and the gel filtration fractions from step 2.19 on an SDS-PAGE and Coomassie stain for analysis. 
	NOTE: It is advisable to run an SDS-PAGE of samples from steps 2.9, 2.15 and 2.16 prior to gel filtration to confirm expression of the target protein.</li>
</ol>

<ol>
	<li>Baneyx, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+Escherichia+coli.">Recombinant protein expression in Escherichia coli.</a> Current opinion in biotechnology. 10 (5), 411-421 (1999).</li><li>Aricescu, A. R., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+recombinant+glycoproteins+in+mammalian+cells:+towards+an+integrative+approach+to+structural+biology.">Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.</a> Current opinion in structural biology. 23 (3), 345-356 (2013).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397 (8), 3173-3178 (2010).</li><li>Boussif, O., Zanta, M. A., Behr, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+galenics+improve+in+vitro+gene+transfer+with+cationic+molecules+up+to+1000-fold.">Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold.</a> Gene therapy. 3 (12), 1074-1080 (1996).</li><li>Godbey, W. T., Wu, K. K., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+(ethylenimine)+and+its+role+in+gene+delivery.">Poly (ethylenimine) and its role in gene delivery.</a> Journal of Controlled Release. 60 ((2-3)), 149-160 (1999).</li><li>Beljelarskaya, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+systems+for+production+of+recombinant+proteins+in+insect+and+mammalian+cells.">Baculovirus expression systems for production of recombinant proteins in insect and mammalian cells.</a> Molecular Biology. 45 (1), 123-138 (2011).</li><li>Cregg, J. M., Vedvick, T. S., Raschke, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+expression+of+foreign+genes+in+Pichia+pastoris.">Recent advances in the expression of foreign genes in Pichia pastoris.</a> Nature Biotechnology. 11 (8), 905-910 (1993).</li><li>Watson, P. J., Fairall, L., Santos, G. M., Schwabe, J. W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+HDAC3+bound+to+co-repressor+and+inositol+tetraphosphate.">Structure of HDAC3 bound to co-repressor and inositol tetraphosphate.</a> Nature. 481 (7381), 335-340 (2013).</li><li>Millard, C. J., Watson, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I+HDACs+Share+a+Common+Mechanism+of+Regulation+by+Inositol+Phosphates.">Class I HDACs Share a Common Mechanism of Regulation by Inositol Phosphates.</a> Molecular Cell. 51 (1), 57-67 (2013).</li><li>Tom, R., Bisson, L., Durocher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+HEK293-EBNA1+Cells+in+Suspension+with+Linear+PEI+for+Production+of+Recombinant+Proteins.">Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins.</a> CSH protocols. , pdb.prot4977 (2008).</li><li>Durocher, Y., Perret, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+and+high-throughput+recombinant+protein+production+by+transient+transfection+of+suspension-growing+human+293-EBNA1+cells.">High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.</a> Nucleic Acids Research. 30 (2), (2002).</li><li>Baldi, L., Muller, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+Gene+Expression+in+Suspension+HEK&#8208;293+Cells:+Application+to+Large&#8208;Scale+Protein+Production.">Transient Gene Expression in Suspension HEK&#8208;293 Cells: Application to Large&#8208;Scale Protein Production.</a> Biotechnology Progress. 21 (1), 148-153 (2005).</li></ol>

The authors declare that they have no competing financial interests.

We have developed a straightforward and cost effective method (PEI costs much less than commercially available lipophilic transfection reagents) for expressing and purifying large amounts of recombinant proteins and multi-subunit complexes from mammalian cells. Optimal transfection and expression efficiency can be reached if highly pure plasmid DNA (260/280 between 1.8 and 2.0) is used in combination with PEI as described in the protocol section. Cells must be cultured in serum- and antibiotic-free media, therefore, sterile technique is strictly required for passaging and transfecting cells in order to avoid costly and time-consuming infections. Cell viability should be 90% or higher and cultures should not be grown to densities greater than 2.5 x 106 cells/ml immediately before a transfection, as doing so will reduce protein yield. For a successful transfection, cultures should only contain single or dividing cells. Clusters may be broken up by vigorous vortexing 20 to 30 sec (Figure 5). The ratio of each plasmid used in the co-transfection can be varied by the user according to the stoichiometry of the complex being studied. Expression efficiency can be optimized for the protein of interest, so that a suitable expression time can be established. Purification should be performed keeping the protein sample cold at all times to reduce the risk of unwanted proteolytic degradation. The choice of tags and purification buffers is critical, since they may interfere with structural elements or active sites of certain proteins, often resulting in reduced solubility and/or loss of enzymatic activity. Small scale transfections are particularly useful for testing different constructs for the purification of large protein complexes.
Today, a large variety of alternative methodologies are available for the expression of recombinant eukaryotic proteins (Table 2). For example, Baculovirus expression in insect cells is widely used due to its high transduction efficiency and its lack of cytotoxicity in comparison to other viral species6. However, the process of making the virus is time-consuming and its instability does not allow the virus to be stored for long periods. On the other hand, yeast expression systems offer the possibility of growing cells to very high densities in fermenter cultures, resulting in high protein yields7. But they still lack the full variety of post-translational modifications required for eukaryotic proteins to fold correctly and interact with their protein partners. HDAC3, for example, is expressed but does not fold in E. coli. However, when expressed in 293F cells, we were able to purify an active enzyme in complex with its co-repressor (SMRT) and a molecule of inositol tetraphosphate (IP4)8, which is not found in prokaryotic cells. This method has also allowed us to purify and solve the crystal structure of HDAC1 interacting with MTA1 in the NuRD complex, which is similarly activated by IP49. Ideally, we would always like to express eukaryotic proteins in their natural, physiological environment. Mammalian expression systems employing HEK 293-EBNA1 cells in bioreactors10-12 have been described and yield very high levels of protein, but these can be complex to use.
Our method is a simple and accessible alternative to expression in bacteria, yeast and bioreactor systems. The expression method is fast and does not require the use of expensive equipment, particularly if the roller bottle or flask atmosphere is replaced with 5% CO2 and standard shaking incubators are used. We have used these protocols to co-transfect multiple plasmids and purify complexes with up to five proteins with yields of &#62;1 mg/L&#160;of culture. Interestingly, the system helps the identification of stable well-behaved complexes. For instance, expression of the binary complex of HDAC1 and Sin3A gave limited yields of protein, yet addition of SDS3 resulted in 5-fold higher amounts and hence guides the structural biologist in choice of suitable constructs and stable complexes for crystallization.

The rapid progress of molecular cell biology and the constant need for improved drugs in medicine has created a need for structural biologists to look at increasingly more complex protein structures. These can often require particular post-translational modifications, molecular chaperones and co-factors to support their elaborate folding and enzymatic activity. Whilst the vast majority of structures present in the Protein Data Bank have been obtained using bacterial expression systems, prokaryotes are unable to perform a large number of these modifications and lack many essential eukaryotic co-factors. This can be an issue for the study of large multi-subunit complexes that are activated by small signaling molecules as well as for nuclear, cell surface and secreted proteins that require elaborate folding machineries. A number of E. coli strains have been engineered to overcome some of these limitations1. In recent years, however, the use of mammalian expression systems has been increasing because they reliably produce eukaryotic proteins that are otherwise problematic to express in other systems2. Today it is possible to obtain stable and transient mammalian expression cell lines through a variety of techniques that range from viral transduction to chemical-mediated transfection, physical gene-transfer such as electroporation and direct injection3. While all of these methods have their own set of advantages and disadvantages, only a few of them are suitable for structural studies being either too expensive and/or time consuming.
Here we describe a very simple, fast, inexpensive yet highly efficient method for expressing protein complexes for structural biology in suspension grown mammalian cells. The approach uses transient co-transfection of a Human Embryonic Kidney (HEK) cell line (e.g., Freestyle HEK 293F cells). These cells have been derived from the HEK 293 cell line, are adapted to grow in suspension cultures, reaching high densities using serum-free media (such as FreeStyle 293 Expression Medium). Cells are then transiently transfected using a branched version of polyethylenimine (PEI), an inexpensive polymeric reagent that has been reported to function for a large range of mammalian cells4 by forming DNA/PEI complexes that enter the host cell by endocytosis5. This method is suitable for both small scale (30 ml) and large-scale (up to 300 ml) experiments and can produce high levels of purified protein complexes. It is particularly useful for studying proteins that require complex folding machineries, co-factors or particular post-translational modifications that cannot be performed by bacteria, yeast and insect cells.
In this protocol we present the expression and purification of the three-protein central scaffold from the Sin3A transcriptional repression complex. This consists of Histone Deacetylase 1 (HDAC1), Suppressor of Defective Silencing 3 (SDS3) and Switch-independent 3 (Sin3A). The purified complex is used for high-throughput crystallization trials.

<table border="0" cellpadding="0" cellspacing="0" width="865">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Reagent/ Equipment</td>
			<td style="width:140px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FreeStyle HEK 293F cells&#160;</td>
			<td style="width:140px;">LifeTechnologies</td>
			<td>R790-07</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:315px;">FreeStyle 293 Expression Medium</td>
			<td style="width:140px;">LifeTechnologies</td>
			<td style="width:119px;">12338-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Anti-FLAG M2 Affinity Gel</td>
			<td style="width:140px;">SIGMA</td>
			<td style="width:119px;">A220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">250 ml Erlenmeyer Flask with Vented Cap</td>
			<td style="width:140px;">Corning</td>
			<td style="width:119px;">431144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Roller bottles with vented caps</td>
			<td style="width:140px;">Corning</td>
			<td style="width:119px;">01836-02</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Polyethylinamine, 25 kDa, branched</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">408727</td>
			<td style="width:292px;">Make 0.5 mg/ml stocks in H2O, adjust pH to 7.0 with dilute HCl and store at -20 - 4&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mammalian expression vector</td>
			<td style="width:140px;">-</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">0.22 &#181;m Centrifugal Filter Units</td>
			<td style="width:140px;">Amicon</td>
			<td style="width:119px;">UFC30GV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">15 ml Ultra centrifugal filters (10 kDa cut-off)</td>
			<td style="width:140px;">Amicon</td>
			<td style="width:119px;">UFC901008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Superdex&#160; 10/300 GL</td>
			<td style="width:140px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-5175-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Here we show a 2 L (8 x 250 ml cultures) transient co-transfection and purification of the HDAC1, SDS3, Sin3A ternary complex. HDAC1 and SDS3 interact with Sin3A through the HDAC-interaction domain (HID) of Sin3A. A typical purification yields up to 1 mg of complex per liter of culture.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig1highres.jpg" width="500" /> 
Figure 1. Schematic of the modified pcDNA 3.1 expression vector. The plasmid was digested with EcoRV and EcoRI to include the affinity-tag and TEV cleavage site on the amino terminus of Sin3A. To clone untagged versions of HDAC1 and SDS3, the vector was digested with KpnI and EcoRI.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig2highres.jpg" width="500" /> 
Figure 2. SDS-PAGE showing the first step of the purification. The &#8220;bound protein&#8221; lane shows the complex bound to the affinity resin. Following TEV digestion, the tag present on Sin3A-HID is cleaved off and the complex is eluted from the resin as shown in the second and third lanes of the gel.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig3highres.jpg" width="500" /> 
Figure 3. Chromatogram of protein-complex purified using size exclusion chromatography. Note that the pure complex eluted in the void volume because it forms a dimer in solution.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig4highres.jpg" width="500" /> 
Figure 4. SDS-PAGE showing the fractions of the gel filtration shown in Figure 3.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51897/51897fig5highres.jpg" width="500" /> 
Figure 5. Trypan blue-stained HEK 293F cells at 2.3x106 cells/ml ready to be transfected. Cells should only be present as single or dividing cells, while large clusters may be broken up by vigorous vortexing for approximately 25 sec.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Possible causes</td>
			<td>Action</td>
		</tr>
		<tr>
			<td rowspan="4">Low protein yield.</td>
			<td>Low number of cells transfected.</td>
			<td>Make sure cell density is approximately 1.0 x 106 before adding the transfection reaction mixture to the culture.</td>
		</tr>
		<tr>
			<td>Cells might have been subcultured too many times.</td>
			<td>Use fresh stock cells after approximately 90 passages.</td>
		</tr>
		<tr>
			<td>DNA may be degraded or have a high amount of impurities.</td>
			<td>Make sure the plasmid DNA being used has a 260/280 ratio between 1.8 and 2.0. Running the DNA on an agarose gel is advisable to assess its quality.</td>
		</tr>
		<tr>
			<td>Protein(s) being expressed are not stable or soluble enough.</td>
			<td>Test different constructs in small scale prior to up-scaling the transfections. Make sure tags do not interfere with the structure of the protein(s) of interest.</td>
		</tr>
		<tr>
			<td>Cells look cloudy and have an unusual color and/or odor.</td>
			<td>Cells are infected with bacteria or yeast.</td>
			<td>Good sterile technique must be used at all times. Fumigating the laminar flow hoods and UV-disinfecting the cell culture room in case of infection helps containing the problem.</td>
		</tr>
		<tr>
			<td rowspan="2">Cells have low viability.</td>
			<td>Wrong pH in media.</td>
			<td>Make sure cultures are grown at 5-8% CO2 at all times.</td>
		</tr>
		<tr>
			<td>Cells have been cultured up to a density over 3.0x106 &#160;cells/ml.</td>
			<td>Cells should not be grown up to densities above 2.5 x 106 cells/ml immediately before a transfection and never above 3.0 x 106 cells/ml.</td>
		</tr>
		<tr>
			<td rowspan="2">Affinity purification did not work</td>
			<td>Protein(s) is not being expressed.</td>
			<td>See above.</td>
		</tr>
		<tr>
			<td>Purification conditions might be wrong.</td>
			<td>Adjust buffer conditions (e.g., high salt, low salt, pH.)</td>
		</tr>
	</tbody>
</table>
Table 1. Troubleshooting.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>System</td>
			<td>Advantages</td>
			<td>Disadvantages</td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>
			<ul>
				<li>Fast expression system</li>
				<li>High protein yields</li>
				<li>Inexpensive</li>
				<li>Suitable for the affordable production of isotopically labeled proteins</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>Lack of important chaperones that may be required for protein folding</li>
				<li>Lack of many post translational modifications</li>
				<li>May lack co-factors that are important for protein function and/or complex assembly.</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>P. pastoris</td>
			<td>
			<ul>
				<li>Can perform the majority of post translational modifications</li>
				<li>Inexpensive</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>Lack some post-translational modifications</li>
				<li>May lack co-factors that are important for protein function and/or complex assembly.</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>Baculovirus expression in insect cells</td>
			<td>
			<ul>
				<li>Can perform all post translational modifications</li>
				<li>Baculovirus is less cytotoxic than other viral strains</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>Preparing the virus for the transduction is time consuming</li>
				<li>The virus is not stable and cannot be stored for long periods of time</li>
				<li>Insect cells may lack co-factors that are important for protein function and/or complex assembly.</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>Bioreactors with mammalian systems</td>
			<td>
			<ul>
				<li>Can perform all post translational modifications</li>
				<li>Can provide all co-factors needed for protein function and complex assembly</li>
				<li>Can culture cells at very high densities</li>
				<li>Can express all mammalian proteins that require complex folding machineries</li>
				<li>Very high protein yields</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>Requires specialized staff to use the bioreactor.</li>
				<li>Expensive equipment is required.</li>
				<li>Optimizing expression conditions may be time consuming.</li>
			</ul>
			</td>
		</tr>
		<tr>
			<td>HEK 293F suspension cells</td>
			<td>
			<ul>
				<li>Can perform all post translational modifications</li>
				<li>Can provide all co-factors needed for protein function and complex assembly</li>
				<li>Can express all mammalian proteins that require complex folding machinery</li>
				<li>Fast and intuitive transfection protocol</li>
				<li>Relatively inexpensive compared to bioreactors and/or other commercially available transfection reagents.</li>
			</ul>
			</td>
			<td>
			<ul>
				<li>Although affordable, not as cheap as expression in bacteria</li>
			</ul>
			</td>
		</tr>
	</tbody>
</table>
Table 2. Advantages and disadvantages of main expression systems.

Watch this Scientific Journal Video about Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach at JoVE.com

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

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 ...

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Research

JoVE Journal

Biology

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

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 an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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 Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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.

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...