{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T20:19:46Z","timestamp":1787170786296,"version":"build-2736575974"},"reference-count":32,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2021,4,29]],"date-time":"2021-04-29T00:00:00Z","timestamp":1619654400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003762","name":"Institut Pasteur","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003762","id-type":"DOI","asserted-by":"crossref"}]},{"name":"CNRS"},{"name":"Sorbonnes University"},{"name":"ANR","award":["ANR-17-CE11-0005-01"],"award-info":[{"award-number":["ANR-17-CE11-0005-01"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome\u2019s transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex\n                    <jats:italic>in vitro<\/jats:italic>\n                    . However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in\n                    <jats:italic>E<\/jats:italic>\n                    .\n                    <jats:italic>coli<\/jats:italic>\n                    , using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)\n                    <jats:sub>2<\/jats:sub>\n                    (nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0250610","type":"journal-article","created":{"date-parts":[[2021,4,29]],"date-time":"2021-04-29T13:29:51Z","timestamp":1619702991000},"page":"e0250610","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":19,"title":["Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5412-8822","authenticated-orcid":true,"given":"Cl\u00e9ment","family":"Madru","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayten Dizkirici","family":"Tekpinar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandrine","family":"Rosario","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3438-4722","authenticated-orcid":true,"given":"Dariusz","family":"Czernecki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8797-6748","authenticated-orcid":true,"given":"S\u00e9bastien","family":"Br\u00fbl\u00e9","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ludovic","family":"Sauguet","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Delarue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,4,29]]},"reference":[{"issue":"10223","key":"pone.0250610.ref001","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0140-6736(20)30183-5","article-title":"Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China","volume":"395","author":"C Huang","year":"2020","journal-title":"The Lancet"},{"issue":"7798","key":"pone.0250610.ref002","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A pneumonia outbreak associated with a new coronavirus of probable bat origin","volume":"579","author":"P Zhou","year":"2020","journal-title":"Nature"},{"issue":"7798","key":"pone.0250610.ref003","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/s41586-020-2008-3","article-title":"A new coronavirus associated with human respiratory disease in China","volume":"579","author":"F Wu","year":"2020","journal-title":"Nature"},{"issue":"5","key":"pone.0250610.ref004","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1016\/S1473-3099(20)30120-1","article-title":"An interactive web-based dashboard to track COVID-19 in real time","volume":"20","author":"E Dong","year":"2020","journal-title":"Lancet Infect Dis"},{"issue":"1","key":"pone.0250610.ref005","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1016\/j.antiviral.2013.08.015","article-title":"From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses","volume":"100","author":"R Hilgenfeld","year":"2013","journal-title":"Antiviral Res"},{"key":"pone.0250610.ref006","first-page":"1","volume-title":"Advances in Virus Research","author":"BW Neuman","year":"2016"},{"key":"pone.0250610.ref007","first-page":"59","volume-title":"Advances in Virus Research","author":"EJ Snijder","year":"2016"},{"key":"pone.0250610.ref008","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.virusres.2017.01.023","article-title":"Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes","volume":"234","author":"CC Posthuma","year":"2017","journal-title":"Virus Res"},{"issue":"396","key":"pone.0250610.ref009","doi-asserted-by":"crossref","first-page":"eaal3653","DOI":"10.1126\/scitranslmed.aal3653","article-title":"Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses","volume":"9","author":"TP Sheahan","year":"2017","journal-title":"Sci Transl Med"},{"issue":"1","key":"pone.0250610.ref010","doi-asserted-by":"crossref","DOI":"10.1186\/s12967-020-02355-3","article-title":"SARS-CoV-2 RNA polymerase as target for antiviral therapy","volume":"18","author":"L Buonaguro","year":"2020","journal-title":"J Transl Med"},{"issue":"1","key":"pone.0250610.ref011","doi-asserted-by":"crossref","DOI":"10.1038\/s41467-020-18463-z","article-title":"Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis","volume":"11","author":"A Shannon","year":"2020","journal-title":"Nat Commun"},{"key":"pone.0250610.ref012","doi-asserted-by":"crossref","first-page":"204020661876448","DOI":"10.1177\/2040206618764483","article-title":"Nucleosides for the treatment of respiratory RNA virus infections","volume":"26","author":"PC Jordan","year":"2018","journal-title":"Antivir Chem Chemother"},{"key":"pone.0250610.ref013","doi-asserted-by":"crossref","first-page":"117477","DOI":"10.1016\/j.lfs.2020.117477","article-title":"Anti-HCV, nucleotide inhibitors, repurposing against COVID-19","volume":"248","author":"AA Elfiky","year":"2020","journal-title":"Life Sci"},{"key":"pone.0250610.ref014","first-page":"247255522094212","article-title":"RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery","author":"W Zhu","year":"2020","journal-title":"SLAS Discov Adv Sci Drug Discov"},{"issue":"7594","key":"pone.0250610.ref015","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1038\/nature17180","article-title":"Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys","volume":"531","author":"TK Warren","year":"2016","journal-title":"Nature"},{"key":"pone.0250610.ref016","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.antiviral.2018.03.003","article-title":"Favipiravir as a potential countermeasure against neglected and emerging RNA viruses","volume":"153","author":"L Delang","year":"2018","journal-title":"Antiviral Res"},{"key":"pone.0250610.ref017","article-title":"Remdesivir for the Treatment of Covid-19\u2014Final Report","author":"JH Beigel","year":"2020","journal-title":"N Engl J Med"},{"key":"pone.0250610.ref018","article-title":"Role of favipiravir in the treatment of COVID-19","author":"S Joshi","year":"2020","journal-title":"Int J Infect Dis"},{"issue":"37","key":"pone.0250610.ref019","doi-asserted-by":"crossref","first-page":"E3900","DOI":"10.1073\/pnas.1323705111","article-title":"One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities","volume":"111","author":"L Subissi","year":"2014","journal-title":"Proc Natl Acad Sci"},{"issue":"1","key":"pone.0250610.ref020","doi-asserted-by":"crossref","DOI":"10.1038\/s41467-019-10280-3","article-title":"Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors","volume":"10","author":"RN Kirchdoerfer","year":"2019","journal-title":"Nat Commun"},{"issue":"6492","key":"pone.0250610.ref021","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1126\/science.abb7498","article-title":"Structure of the RNA-dependent RNA polymerase from COVID-19 virus","volume":"368","author":"Y Gao","year":"2020","journal-title":"Science"},{"issue":"11","key":"pone.0250610.ref022","doi-asserted-by":"crossref","first-page":"107774","DOI":"10.1016\/j.celrep.2020.107774","article-title":"Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2","volume":"31","author":"Q Peng","year":"2020","journal-title":"Cell Rep"},{"key":"pone.0250610.ref023","article-title":"Structure of replicating SARS-CoV-2 polymerase","author":"HS Hillen","year":"2020","journal-title":"Nature"},{"issue":"2","key":"pone.0250610.ref024","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/j.cell.2020.05.034","article-title":"Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase","volume":"182","author":"Q Wang","year":"2020","journal-title":"Cell"},{"issue":"1","key":"pone.0250610.ref025","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1038\/s41467-020-20542-0","article-title":"Mechanism of SARS-CoV-2 polymerase stalling by remdesivir","volume":"12","author":"G Kokic","year":"2021","journal-title":"Nat Commun."},{"issue":"6498","key":"pone.0250610.ref026","doi-asserted-by":"crossref","first-page":"1499","DOI":"10.1126\/science.abc1560","article-title":"Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir","volume":"368","author":"W Yin","year":"2020","journal-title":"Science"},{"issue":"7","key":"pone.0250610.ref027","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2021946118","article-title":"Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence of favipiravir-RTP","volume":"118","author":"K Naydenova","year":"2021","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"6","key":"pone.0250610.ref028","first-page":"1560","article-title":"Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2","volume":"182","author":"J Chen","year":"2020","journal-title":"Replication-Transcription Complex. Cell"},{"issue":"1","key":"pone.0250610.ref029","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.cell.2020.11.016","article-title":"Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis","volume":"184","author":"L Yan","year":"2021","journal-title":"Cell"},{"issue":"1","key":"pone.0250610.ref030","doi-asserted-by":"crossref","first-page":"5874","DOI":"10.1038\/s41467-020-19770-1","article-title":"Architecture of a SARS-CoV-2 mini replication and transcription complex","volume":"11","author":"L Yan","year":"2020","journal-title":"Nat Commun"},{"issue":"2","key":"pone.0250610.ref031","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1016\/S0006-3495(02)75469-6","article-title":"Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems","volume":"82","author":"P Schuck","year":"2002","journal-title":"Biophys J"},{"issue":"1","key":"pone.0250610.ref032","doi-asserted-by":"crossref","first-page":"100357","DOI":"10.1016\/j.xpro.2021.100357","article-title":"Expression and purification of tag-free SARS-CoV-2 RNA-dependent RNA polymerase in Escherichia coli","volume":"2","author":"TL Dangerfield","year":"2021","journal-title":"STAR Protoc"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0250610","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,4,29]],"date-time":"2021-04-29T13:30:29Z","timestamp":1619703029000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0250610"}},"subtitle":[],"editor":[{"given":"Filippo","family":"Prischi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2021,4,29]]},"references-count":32,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2021,4,29]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0250610","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,29]]}}}