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><tbody><tr><td>FreeStyle HEK 293F cells&amp;nbsp;</td><td>LifeTechnologies</td><td>R790-07</td><td></td></tr><tr><td>FreeStyle 293 Expression Medium</td><td>LifeTechnologies</td><td>12338-018</td><td></td></tr><tr><td>Anti-FLAG M2 Affinity Gel</td><td>SIGMA</td><td>A220</td><td></td></tr><tr><td>250 ml Erlenmeyer Flask with Vented Cap</td><td>Corning</td><td>431144</td><td></td></tr><tr><td>Roller bottles with vented caps</td><td>Corning</td><td>01836-02</td><td></td></tr><tr><td>Polyethylenimine, 25 kDa, branched</td><td>Sigma-Aldrich</td><td>408727</td><td>Make 0.5 mg/ml stocks in H&lt;sub&gt;2&lt;/sub&gt;O, adjust pH to 7.0 with dilute HCl and store at -20 - 4&amp;ordm;C</td></tr><tr><td>Mammalian expression vector</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>0.22 &amp;micro;m Centrifugal Filter Units</td><td>Amicon</td><td>UFC30GV00</td><td></td></tr><tr><td>15 ml Ultra centrifugal filters (10 kDa cut-off)</td><td>Amicon</td><td>UFC901008</td><td></td></tr><tr><td>Superdex&amp;nbsp; 10/300 GL</td><td>GE Healthcare Life Sciences</td><td>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.

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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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Biology

53.7K Views.  University of Leicester. 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.

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

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Genetics

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Bioengineering

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

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) ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunology and Infection

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. ...

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 ...

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

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

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

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

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

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

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

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

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.

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

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