Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins
We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in viv...
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| creator | Zhong, Nan Loppnau, Peter Seitova, Alma Ravichandran, Mani Fenner, Maria Jain, Harshika Bhattacharya, Anandi Hutchinson, Ashley Paduch, Marcin Lu, Vincent Olszewski, Michal Kossiakoff, Anthony A Dowdell, Evan Koide, Akiko Koide, Shohei Huang, Haiming Nadeem, Vincent Sidhu, Sachdev S Greenblatt, Jack F Marcon, Edyta Arrowsmith, Cheryl H Edwards, Aled M Gräslund, Susanne |
| description | We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs ( |
| doi_str_mv | 10.1371/journal.pone.0139695 |
| format | Article |
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Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0139695</identifier><identifier>PMID: 26437229</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Antibodies ; Antibody Formation - physiology ; Antigens ; Antigens - immunology ; Biochemistry ; Biomedical research ; Biotinylation ; Chromatin ; Cloning, Molecular ; Comparative analysis ; Consortia ; Cytochrome ; E coli ; Enzymes ; Escherichia coli ; Genetic aspects ; Genomics ; Humans ; Immunoglobulin Fab Fragments - immunology ; Immunoglobulin G ; Immunoglobulins ; Immunoprecipitation ; In vivo methods and tests ; Lysates ; Mammalian cells ; Mammals ; Molecular biology ; Peptide Library ; Peptides ; Phage display ; Phages ; Physiological aspects ; Proteins ; Proteomics ; Recombinant Proteins - immunology ; RNA polymerase ; Stem cells ; Workflow ; Yield</subject><ispartof>PloS one, 2015-10, Vol.10 (10), p.e0139695-e0139695</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Zhong et al 2015 Zhong et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-61f6f95bf35bb081fc04d4730b74993e51ecb269948e0b4d4a49b1872c78fe713</citedby><cites>FETCH-LOGICAL-c758t-61f6f95bf35bb081fc04d4730b74993e51ecb269948e0b4d4a49b1872c78fe713</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593582/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593582/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26437229$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhong, Nan</creatorcontrib><creatorcontrib>Loppnau, Peter</creatorcontrib><creatorcontrib>Seitova, Alma</creatorcontrib><creatorcontrib>Ravichandran, Mani</creatorcontrib><creatorcontrib>Fenner, Maria</creatorcontrib><creatorcontrib>Jain, Harshika</creatorcontrib><creatorcontrib>Bhattacharya, Anandi</creatorcontrib><creatorcontrib>Hutchinson, Ashley</creatorcontrib><creatorcontrib>Paduch, Marcin</creatorcontrib><creatorcontrib>Lu, Vincent</creatorcontrib><creatorcontrib>Olszewski, Michal</creatorcontrib><creatorcontrib>Kossiakoff, Anthony A</creatorcontrib><creatorcontrib>Dowdell, Evan</creatorcontrib><creatorcontrib>Koide, Akiko</creatorcontrib><creatorcontrib>Koide, Shohei</creatorcontrib><creatorcontrib>Huang, Haiming</creatorcontrib><creatorcontrib>Nadeem, Vincent</creatorcontrib><creatorcontrib>Sidhu, Sachdev S</creatorcontrib><creatorcontrib>Greenblatt, Jack F</creatorcontrib><creatorcontrib>Marcon, Edyta</creatorcontrib><creatorcontrib>Arrowsmith, Cheryl H</creatorcontrib><creatorcontrib>Edwards, Aled M</creatorcontrib><creatorcontrib>Gräslund, Susanne</creatorcontrib><title>Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. 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Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins</title><author>Zhong, Nan ; Loppnau, Peter ; Seitova, Alma ; Ravichandran, Mani ; Fenner, Maria ; Jain, Harshika ; Bhattacharya, Anandi ; Hutchinson, Ashley ; Paduch, Marcin ; Lu, Vincent ; Olszewski, Michal ; Kossiakoff, Anthony A ; Dowdell, Evan ; Koide, Akiko ; Koide, Shohei ; Huang, Haiming ; Nadeem, Vincent ; Sidhu, Sachdev S ; Greenblatt, Jack F ; Marcon, Edyta ; Arrowsmith, Cheryl H ; Edwards, Aled M ; Gräslund, Susanne</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c758t-61f6f95bf35bb081fc04d4730b74993e51ecb269948e0b4d4a49b1872c78fe713</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Antibodies</topic><topic>Antibody Formation - physiology</topic><topic>Antigens</topic><topic>Antigens - immunology</topic><topic>Biochemistry</topic><topic>Biomedical 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one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zhong, Nan</au><au>Loppnau, Peter</au><au>Seitova, Alma</au><au>Ravichandran, Mani</au><au>Fenner, Maria</au><au>Jain, Harshika</au><au>Bhattacharya, Anandi</au><au>Hutchinson, Ashley</au><au>Paduch, Marcin</au><au>Lu, Vincent</au><au>Olszewski, Michal</au><au>Kossiakoff, Anthony A</au><au>Dowdell, Evan</au><au>Koide, Akiko</au><au>Koide, Shohei</au><au>Huang, Haiming</au><au>Nadeem, Vincent</au><au>Sidhu, Sachdev S</au><au>Greenblatt, Jack F</au><au>Marcon, Edyta</au><au>Arrowsmith, Cheryl H</au><au>Edwards, Aled M</au><au>Gräslund, Susanne</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-10-05</date><risdate>2015</risdate><volume>10</volume><issue>10</issue><spage>e0139695</spage><epage>e0139695</epage><pages>e0139695-e0139695</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26437229</pmid><doi>10.1371/journal.pone.0139695</doi><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_1719374548 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Antibodies Antibody Formation - physiology Antigens Antigens - immunology Biochemistry Biomedical research Biotinylation Chromatin Cloning, Molecular Comparative analysis Consortia Cytochrome E coli Enzymes Escherichia coli Genetic aspects Genomics Humans Immunoglobulin Fab Fragments - immunology Immunoglobulin G Immunoglobulins Immunoprecipitation In vivo methods and tests Lysates Mammalian cells Mammals Molecular biology Peptide Library Peptides Phage display Phages Physiological aspects Proteins Proteomics Recombinant Proteins - immunology RNA polymerase Stem cells Workflow Yield |
| title | Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-02T15%3A16%3A04IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Optimizing%20Production%20of%20Antigens%20and%20Fabs%20in%20the%20Context%20of%20Generating%20Recombinant%20Antibodies%20to%20Human%20Proteins&rft.jtitle=PloS%20one&rft.au=Zhong,%20Nan&rft.date=2015-10-05&rft.volume=10&rft.issue=10&rft.spage=e0139695&rft.epage=e0139695&rft.pages=e0139695-e0139695&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0139695&rft_dat=%3Cgale_plos_%3EA430767660%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1719374548&rft_id=info:pmid/26437229&rft_galeid=A430767660&rft_doaj_id=oai_doaj_org_article_dd18dddf47c540eb9b460223ee62eb80&rfr_iscdi=true |