A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells
We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-S...
Gespeichert in:
| Veröffentlicht in: | Protein expression and purification 2008-11, Vol.62 (1), p.83-89 |
|---|---|
| Hauptverfasser: | , , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 89 |
|---|---|
| container_issue | 1 |
| container_start_page | 83 |
| container_title | Protein expression and purification |
| container_volume | 62 |
| creator | Nettleship, Joanne E. Ren, Jingshan Rahman, Nahid Berrow, Nick S. Hatherley, Deborah Neil Barclay, A. Owens, Raymond J. |
| description | We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPγ). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion™ PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4
mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3
Å and 2.4
Å, respectively. |
| doi_str_mv | 10.1016/j.pep.2008.06.017 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_69673846</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S1046592808001745</els_id><sourcerecordid>69673846</sourcerecordid><originalsourceid>FETCH-LOGICAL-c382t-7dc7755c30eb733c69d0611c14c62d0136ef5cd2390f061d6f9b7c8c1dde32ab3</originalsourceid><addsrcrecordid>eNqFkU1v1DAQhi1URD_gB3BBPvWWMLY3diJOVbXQikpcytlK7EnxKpsEj0Pbf4_DrsStPXkkP_PKfh_GPgooBQj9eVfOOJcSoC5BlyDMG3YmoNEFSNOcrPNGF1Uj61N2TrQDEEJD9Y6dilpraerqjD1e8TnMOIQReT9Fnn4hn-PkF5fCNPKp5-2YQjf5Z97H9mGPY6J_IKWYmSW2Qx4XH5D4QmF84Cm2I4XMcXyaIxKtOWHkN9vvXDbqnjscBnrP3vbtQPjheF6wn1-399c3xd2Pb7fXV3eFU7VMhfHOmKpyCrAzSjndeNBCOLFxWnoQSmNfOS9VA32-8LpvOuNqJ7xHJdtOXbDLQ27-1O8FKdl9oPUF7YjTQlY32qh6o18FZS5TVFWdQXEAXZyIIvZ2jmHfxmcrwK5a7M5mLXbVYkHbrCXvfDqGL90e_f-No4cMfDkAmLv4EzBacrlDhz5EdMn6KbwQ_xcPN575</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>21041558</pqid></control><display><type>article</type><title>A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells</title><source>MEDLINE</source><source>Access via ScienceDirect (Elsevier)</source><creator>Nettleship, Joanne E. ; Ren, Jingshan ; Rahman, Nahid ; Berrow, Nick S. ; Hatherley, Deborah ; Neil Barclay, A. ; Owens, Raymond J.</creator><creatorcontrib>Nettleship, Joanne E. ; Ren, Jingshan ; Rahman, Nahid ; Berrow, Nick S. ; Hatherley, Deborah ; Neil Barclay, A. ; Owens, Raymond J.</creatorcontrib><description>We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPγ). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion™ PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4
mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3
Å and 2.4
Å, respectively.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/j.pep.2008.06.017</identifier><identifier>PMID: 18662785</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Animals ; Antibodies, Monoclonal - biosynthesis ; Antibodies, Monoclonal - chemistry ; Antibodies, Monoclonal - isolation & purification ; Antibody fragments ; Antigens, Surface - immunology ; Antigens, Surface - metabolism ; Base Sequence ; Cells, Cultured ; Crystallography, X-Ray ; Fabs ; Genetic Vectors - genetics ; Genetic Vectors - metabolism ; Immunoglobulin Fab Fragments - biosynthesis ; Immunoglobulin Fab Fragments - chemistry ; Immunoglobulin Fab Fragments - isolation & purification ; Mice ; Models, Molecular ; Molecular Sequence Data ; Protein Conformation ; Receptors, Cell Surface - immunology ; Receptors, Cell Surface - metabolism ; Transient expression ; X-ray crystallography</subject><ispartof>Protein expression and purification, 2008-11, Vol.62 (1), p.83-89</ispartof><rights>2008 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c382t-7dc7755c30eb733c69d0611c14c62d0136ef5cd2390f061d6f9b7c8c1dde32ab3</citedby><cites>FETCH-LOGICAL-c382t-7dc7755c30eb733c69d0611c14c62d0136ef5cd2390f061d6f9b7c8c1dde32ab3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.pep.2008.06.017$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18662785$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nettleship, Joanne E.</creatorcontrib><creatorcontrib>Ren, Jingshan</creatorcontrib><creatorcontrib>Rahman, Nahid</creatorcontrib><creatorcontrib>Berrow, Nick S.</creatorcontrib><creatorcontrib>Hatherley, Deborah</creatorcontrib><creatorcontrib>Neil Barclay, A.</creatorcontrib><creatorcontrib>Owens, Raymond J.</creatorcontrib><title>A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPγ). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion™ PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4
mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3
Å and 2.4
Å, respectively.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Antibodies, Monoclonal - biosynthesis</subject><subject>Antibodies, Monoclonal - chemistry</subject><subject>Antibodies, Monoclonal - isolation & purification</subject><subject>Antibody fragments</subject><subject>Antigens, Surface - immunology</subject><subject>Antigens, Surface - metabolism</subject><subject>Base Sequence</subject><subject>Cells, Cultured</subject><subject>Crystallography, X-Ray</subject><subject>Fabs</subject><subject>Genetic Vectors - genetics</subject><subject>Genetic Vectors - metabolism</subject><subject>Immunoglobulin Fab Fragments - biosynthesis</subject><subject>Immunoglobulin Fab Fragments - chemistry</subject><subject>Immunoglobulin Fab Fragments - isolation & purification</subject><subject>Mice</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Protein Conformation</subject><subject>Receptors, Cell Surface - immunology</subject><subject>Receptors, Cell Surface - metabolism</subject><subject>Transient expression</subject><subject>X-ray crystallography</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhi1URD_gB3BBPvWWMLY3diJOVbXQikpcytlK7EnxKpsEj0Pbf4_DrsStPXkkP_PKfh_GPgooBQj9eVfOOJcSoC5BlyDMG3YmoNEFSNOcrPNGF1Uj61N2TrQDEEJD9Y6dilpraerqjD1e8TnMOIQReT9Fnn4hn-PkF5fCNPKp5-2YQjf5Z97H9mGPY6J_IKWYmSW2Qx4XH5D4QmF84Cm2I4XMcXyaIxKtOWHkN9vvXDbqnjscBnrP3vbtQPjheF6wn1-399c3xd2Pb7fXV3eFU7VMhfHOmKpyCrAzSjndeNBCOLFxWnoQSmNfOS9VA32-8LpvOuNqJ7xHJdtOXbDLQ27-1O8FKdl9oPUF7YjTQlY32qh6o18FZS5TVFWdQXEAXZyIIvZ2jmHfxmcrwK5a7M5mLXbVYkHbrCXvfDqGL90e_f-No4cMfDkAmLv4EzBacrlDhz5EdMn6KbwQ_xcPN575</recordid><startdate>20081101</startdate><enddate>20081101</enddate><creator>Nettleship, Joanne E.</creator><creator>Ren, Jingshan</creator><creator>Rahman, Nahid</creator><creator>Berrow, Nick S.</creator><creator>Hatherley, Deborah</creator><creator>Neil Barclay, A.</creator><creator>Owens, Raymond J.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20081101</creationdate><title>A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells</title><author>Nettleship, Joanne E. ; Ren, Jingshan ; Rahman, Nahid ; Berrow, Nick S. ; Hatherley, Deborah ; Neil Barclay, A. ; Owens, Raymond J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c382t-7dc7755c30eb733c69d0611c14c62d0136ef5cd2390f061d6f9b7c8c1dde32ab3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Antibodies, Monoclonal - biosynthesis</topic><topic>Antibodies, Monoclonal - chemistry</topic><topic>Antibodies, Monoclonal - isolation & purification</topic><topic>Antibody fragments</topic><topic>Antigens, Surface - immunology</topic><topic>Antigens, Surface - metabolism</topic><topic>Base Sequence</topic><topic>Cells, Cultured</topic><topic>Crystallography, X-Ray</topic><topic>Fabs</topic><topic>Genetic Vectors - genetics</topic><topic>Genetic Vectors - metabolism</topic><topic>Immunoglobulin Fab Fragments - biosynthesis</topic><topic>Immunoglobulin Fab Fragments - chemistry</topic><topic>Immunoglobulin Fab Fragments - isolation & purification</topic><topic>Mice</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Protein Conformation</topic><topic>Receptors, Cell Surface - immunology</topic><topic>Receptors, Cell Surface - metabolism</topic><topic>Transient expression</topic><topic>X-ray crystallography</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nettleship, Joanne E.</creatorcontrib><creatorcontrib>Ren, Jingshan</creatorcontrib><creatorcontrib>Rahman, Nahid</creatorcontrib><creatorcontrib>Berrow, Nick S.</creatorcontrib><creatorcontrib>Hatherley, Deborah</creatorcontrib><creatorcontrib>Neil Barclay, A.</creatorcontrib><creatorcontrib>Owens, Raymond J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nettleship, Joanne E.</au><au>Ren, Jingshan</au><au>Rahman, Nahid</au><au>Berrow, Nick S.</au><au>Hatherley, Deborah</au><au>Neil Barclay, A.</au><au>Owens, Raymond J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2008-11-01</date><risdate>2008</risdate><volume>62</volume><issue>1</issue><spage>83</spage><epage>89</epage><pages>83-89</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPγ). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion™ PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4
mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3
Å and 2.4
Å, respectively.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>18662785</pmid><doi>10.1016/j.pep.2008.06.017</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1046-5928 |
| ispartof | Protein expression and purification, 2008-11, Vol.62 (1), p.83-89 |
| issn | 1046-5928 1096-0279 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_69673846 |
| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | Amino Acid Sequence Animals Antibodies, Monoclonal - biosynthesis Antibodies, Monoclonal - chemistry Antibodies, Monoclonal - isolation & purification Antibody fragments Antigens, Surface - immunology Antigens, Surface - metabolism Base Sequence Cells, Cultured Crystallography, X-Ray Fabs Genetic Vectors - genetics Genetic Vectors - metabolism Immunoglobulin Fab Fragments - biosynthesis Immunoglobulin Fab Fragments - chemistry Immunoglobulin Fab Fragments - isolation & purification Mice Models, Molecular Molecular Sequence Data Protein Conformation Receptors, Cell Surface - immunology Receptors, Cell Surface - metabolism Transient expression X-ray crystallography |
| title | A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T12%3A32%3A08IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20pipeline%20for%20the%20production%20of%20antibody%20fragments%20for%20structural%20studies%20using%20transient%20expression%20in%20HEK%20293T%20cells&rft.jtitle=Protein%20expression%20and%20purification&rft.au=Nettleship,%20Joanne%20E.&rft.date=2008-11-01&rft.volume=62&rft.issue=1&rft.spage=83&rft.epage=89&rft.pages=83-89&rft.issn=1046-5928&rft.eissn=1096-0279&rft_id=info:doi/10.1016/j.pep.2008.06.017&rft_dat=%3Cproquest_cross%3E69673846%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=21041558&rft_id=info:pmid/18662785&rft_els_id=S1046592808001745&rfr_iscdi=true |