This work was supported by an Ontario HIV Treatment Network Research Operating Grant (ROG-G645) and Canadian Institutes of Health Research New Investigator Award (MSH-113554) to JEL, and University of Toronto Fellowships to HA, FCA, and JDC. The authors would like to thank Marnie Fusco, Dafna Abelson and Dr. Erica Ollmann Saphire at The Scripps Research Institute (La Jolla, CA) for providing cells, ebolavirus GP expression vector and general advice.

1. Preparation Work - Constructs and Cell Cultures
Before starting the protocol, the gene of interest should be codon-optimized for expression in mammalian cells, and cloned into an appropriate expression vector using standard molecular biology techniques. In order to ensure the highest chance for successful expression, multiple variants of the gene of interest should be generated. Many mammalian expression vectors are available commercially and have various purification tags (polyhistidine, hem...

<ol>
	<li>Celik, E., Calik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+by+yeast+cells.">Production of recombinant proteins by yeast cells.</a> Biotechnol. Adv. , (2011).</li><li>Yokoyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+expression+systems+for+structural+genomics+and+proteomics.">Protein expression systems for structural genomics and proteomics.</a> Curr. Opin. Chem. Biol. 7, 39-43 (2003).</li><li>Nettleship, J. E., Assenberg, R., Diprose, J. M., Rahman-Huq, N., 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=Recent+advances+in+the+production+of+proteins+in+insect+and+mammalian+cells+for+structural+biology.">Recent advances in the production of proteins in insect and mammalian cells for structural biology.</a> J. Struct. Biol. 172, 55-65 (2010).</li><li>Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis:+Applications+come+of+age.">Cell-free protein synthesis: Applications come of age.</a> Biotechnol. Adv. , (2011).</li><li>Lee, J. E. <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+the+Ebola+virus+glycoprotein+bound+to+an+antibody+from+a+human+survivor.">Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.</a> Nature. 454, 177-182 (2008).</li><li>Bowden, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+Nipah+and+Hendra+virus+attachment+to+their+cell-surface+receptor+ephrin-B2.">Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2.</a> Nat. Struct. Mol. Biol. 15, 567-572 (2008).</li><li>Evans, M. J., Hartman, S. L., Wolff, D. W., Rollins, S. A., Squinto, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+expression+of+an+anti-human+C5+chimeric+Fab+utilizing+a+vector+that+replicates+in+COS+and+293+cells.">Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.</a> J. Immunol. Methods. 184, 123-138 (1995).</li><li>Ye, J. <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+protein+expression+in+scalable+CHO+transient+transfection.">High-level protein expression in scalable CHO transient transfection.</a> Biotechnol. Bioeng. 103, 542-551 (2009).</li><li>Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+protein+therapeutics+in+cultivated+mammalian+cells.">Production of recombinant protein therapeutics in cultivated mammalian cells.</a> Nat Biotechnol. 22, 1393-1398 (2004).</li><li>Lee, J. E., Fusco, M. H., Hessell, A. J., Burton, D. R., Saphire, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+tactics+used+in+determining+the+structure+of+trimeric,+prefusion+Ebola+virus+GP.">Techniques and tactics used in determining the structure of trimeric, prefusion Ebola virus GP.</a> Acta Cryst. 65, 1162-1180 (2009).</li><li>Aricescu, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+expression:+developments+for+structural+proteomics.">Eukaryotic expression: developments for structural proteomics.</a> Acta. Crystallogr. D Biol. Crystallogr. 62, 1114-1124 (2006).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta. Crystallogr. D Biol. Crystallogr. D62, 1243-1250 (2006).</li><li>Chang, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Lee, J. E., Fusco, M. H., Saphire, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+platform+for+screening+expression+and+crystallization+of+glycoproteins+produced+in+human+cells.">An efficient platform for screening expression and crystallization of glycoproteins produced in human cells.</a> Nature Protocols. 4, 592-604 (2009).</li><li>Nettleship, J. E., Rahman-Huq, N., 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=The+production+of+glycoproteins+by+transient+expression+in+Mammalian+cells.">The production of glycoproteins by transient expression in Mammalian cells.</a> Methods Mol. Biol. 498, 245-263 (2009).</li><li>Neil, S. J., Zang, T., Bieniasz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetherin+inhibits+retrovirus+release+and+is+antagonized+by+HIV-1+Vpu.">Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu.</a> Nature. 451, 425-430 (2008).</li><li>Nakamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterodimerization+of+the+IL-2+receptor+beta-+and+gamma-chain+cytoplasmic+domains+is+required+for+signalling.">Heterodimerization of the IL-2 receptor beta- and gamma-chain cytoplasmic domains is required for signalling.</a> Nature. 369, 330-333 (1994).</li><li>Wang, X., Rickert, M., Garcia, K. C. <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+the+quaternary+complex+of+interleukin-2+with+its+alpha,+beta,+and+gammac+receptors.">Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors.</a> Science. 310, 1159-1163 (2005).</li></ol>

The 10-layer cell factories are an effective vessel for the production of milligram quantities of protein. A major advantage of using the cell factory over other traditional vessels, such as roller bottles, shake flasks or spinner flasks, is that they do not require the purchase of any additional laboratory equipment. A standard CO2 incubator (~6.0 cu. ft.) will easily accommodate four 10-layer cell factories (Fig. 2). In addition, these vessels require less labor and space than dishes, flasks...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company </td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Alkaline phosphatase (BCIP/NBT) liquid substrate solution</td>
 <td>Sigma</td>
 <td>B6404</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibiotic/Antimycotic, 100X</td>
 <td>Invitrogen</td>
 <td>15240062</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-HA affinity matrix, clone 3F10</td>
 <td>Roche</td>
 <td>1815016</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-HA murine mAb, clone 16B12</td>
 <td>Covance</td>
 <td>MMS-101P</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture flask, T75 cm2 tissue culture treated</td>
 <td>Corning</td>
 <td>430641</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture flask, T225 cm2 tissue culture treated</td>
 <td>Corning</td>
 <td>431082</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell culture plates,6-well tissue culture treated</td>
 <td>Corning</td>
 <td>3516</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell factory, 10-layer CellSTACK</td>
 <td>Corning</td>
 <td>3312</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centramate Omega 5K Cassette</td>
 <td>Pall</td>
 <td>OS005C12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centramate Omega 30K Cassette</td>
 <td>Pall</td>
 <td>OS030C12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chromatography glass column, 1.0x10 cm</td>
 <td>Kontes</td>
 <td>4204001010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>Sigma</td>
 <td>17850</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CO2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's modified Eagle's media (DMEM)</td>
 <td>Sigma</td>
 <td>D5796</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS), heat inactivated</td>
 <td>Invitrogen</td>
 <td>12484-028</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FuGENE HD transfection reagent</td>
 <td>Promega</td>
 <td>4709691001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GeneJuice transfection reagent</td>
 <td>EMD/Merck</td>
 <td>70967-6</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Sigma</td>
 <td>G8898</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Goat anti-mouse IgG F(ab')2 alkaline</td>
 <td>Thermo Scientific</td>
 <td>31324</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>phosphatase-conjugated antibody</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hemagglutinin (HA) peptide, 100 mg</td>
 <td>Genscript</td>
 <td>custom synthesis</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>(sequence: YPYDVPDYA; 95% purity)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEK 293T cells</td>
 <td>ATCC</td>
 <td>CRL-11268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Household bleach (4% w/v sodium hypochlorite)</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Immobilon-P PVDF membrane</td>
 <td>Millipore</td>
 <td>IPVH07850</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MiniPrep plasmid purification kit, PureLink Quick</td>
 <td>Invitrogen</td>
 <td>K2100-11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MaxiPrep plasmid purification kit, PureLink HiPure</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaN3</td>
 <td>Sigma</td>
 <td>S8032</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pDISPLAY expression vector</td>
 <td>Invitrogen</td>
 <td>V660-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin (pen/strep), 100X</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline (PBS), sterile 1X</td>
 <td>Sigma</td>
 <td>D8537</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethyleneimine (PEI), linear 25 kDa</td>
 <td>Polyscience</td>
 <td>23966</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Skim milk dry powder</td>
 <td>Carnation</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Stericup-GP PES vacuum filtration unit,</td>
 <td>Millipore</td>
 <td>SCGPU05RE</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 &mu;m, 500 ml capacity</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypan blue</td>
 <td>Invitrogen</td>
 <td>15250061</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA, 0.05% (w/v)</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tween-20</td>
 <td>Sigma</td>
 <td>P7949</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Valproic acid</td>
 <td>Sigma</td>
 <td>P4543</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centramate tangential flow system</td>
 <td>Pall</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CO2 humidified incubator, standard 6.0 cu. ft.</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrophoresis and transfer unit</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Incubator, 37 &deg;C</td>
 <td>various brands are available</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

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

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. ...

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21.0K visualizações.  University of Toronto. Na era pós-genômica humana, a disponibilidade de proteínas recombinantes em conformações nativas é crucial para a investigação estrutural, funcional e terapêutico e desenvolvimento. Aqui, nós descrevemos um-teste e em grande escala sistema de expressão de proteínas em células humanas embrionárias de rim 293T que podem ser utilizados para produzir uma variedade de proteínas recombinantes.