High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector
The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-bindin...
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| creator | Sinegubova, Maria V Orlova, Nadezhda A Kovnir, Sergey V Dayanova, Lutsia K Vorobiev, Ivan I |
| description | The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies. |
| doi_str_mv | 10.1371/journal.pone.0242890 |
| format | Article |
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The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0242890</identifier><identifier>PMID: 33529230</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Antibodies ; Antigens ; Bioengineering ; Biology and life sciences ; Biotechnology ; Blood coagulation ; CHO Cells ; Cloning ; Clotting ; Coronaviridae ; Coronaviruses ; COVID-19 ; Cricetulus ; Deoxyribonucleic acid ; DNA ; Editing ; Electronic mail ; Epstein-Barr virus ; Follicle-stimulating hormone ; Gene Expression ; Genetic aspects ; Genetic Vectors ; Glycoproteins ; Glycosylation ; Humans ; Laboratories ; Long terminal repeat ; Mammals ; Medicine and health sciences ; Methodology ; Middle East respiratory syndrome ; Pandemics ; Peptide Elongation Factor 1 - genetics ; Plasmids ; Polymerase chain reaction ; Protein Domains ; Protein S ; Proteins ; Research and Analysis Methods ; Research facilities ; Respiration ; Respiratory diseases ; Serology ; Severe acute respiratory syndrome ; Severe acute respiratory syndrome coronavirus 2 ; Social research ; Spike Glycoprotein, Coronavirus - chemistry ; Spike Glycoprotein, Coronavirus - genetics ; Spike Glycoprotein, Coronavirus - isolation & purification ; Structure ; Transfection - methods ; Vectors (Biology) ; Viral diseases ; Viral proteins ; Virus research ; Viruses ; Yeasts</subject><ispartof>PloS one, 2021-02, Vol.16 (2), p.e0242890-e0242890</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>2021 Sinegubova 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>2021 Sinegubova et al 2021 Sinegubova et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c743t-f7f6f90b5046d5aaa35a5dd64cc4ae230b3cf1cecc25328a95c9c73e1d1fbaac3</citedby><cites>FETCH-LOGICAL-c743t-f7f6f90b5046d5aaa35a5dd64cc4ae230b3cf1cecc25328a95c9c73e1d1fbaac3</cites><orcidid>0000-0001-9483-2892 ; 0000-0003-0064-4458</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853477/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853477/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,725,778,782,862,883,2098,2917,23849,27907,27908,53774,53776,79351,79352</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33529230$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Ho, Paulo Lee</contributor><creatorcontrib>Sinegubova, Maria V</creatorcontrib><creatorcontrib>Orlova, Nadezhda A</creatorcontrib><creatorcontrib>Kovnir, Sergey V</creatorcontrib><creatorcontrib>Dayanova, Lutsia K</creatorcontrib><creatorcontrib>Vorobiev, Ivan I</creatorcontrib><title>High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. 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The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.</description><subject>Animals</subject><subject>Antibodies</subject><subject>Antigens</subject><subject>Bioengineering</subject><subject>Biology and life sciences</subject><subject>Biotechnology</subject><subject>Blood coagulation</subject><subject>CHO Cells</subject><subject>Cloning</subject><subject>Clotting</subject><subject>Coronaviridae</subject><subject>Coronaviruses</subject><subject>COVID-19</subject><subject>Cricetulus</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Editing</subject><subject>Electronic mail</subject><subject>Epstein-Barr virus</subject><subject>Follicle-stimulating hormone</subject><subject>Gene Expression</subject><subject>Genetic aspects</subject><subject>Genetic Vectors</subject><subject>Glycoproteins</subject><subject>Glycosylation</subject><subject>Humans</subject><subject>Laboratories</subject><subject>Long 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expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector</title><author>Sinegubova, Maria V ; Orlova, Nadezhda A ; Kovnir, Sergey V ; Dayanova, Lutsia K ; Vorobiev, Ivan I</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c743t-f7f6f90b5046d5aaa35a5dd64cc4ae230b3cf1cecc25328a95c9c73e1d1fbaac3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Animals</topic><topic>Antibodies</topic><topic>Antigens</topic><topic>Bioengineering</topic><topic>Biology and life sciences</topic><topic>Biotechnology</topic><topic>Blood coagulation</topic><topic>CHO Cells</topic><topic>Cloning</topic><topic>Clotting</topic><topic>Coronaviridae</topic><topic>Coronaviruses</topic><topic>COVID-19</topic><topic>Cricetulus</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Editing</topic><topic>Electronic 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one</jtitle><addtitle>PLoS One</addtitle><date>2021-02-02</date><risdate>2021</risdate><volume>16</volume><issue>2</issue><spage>e0242890</spage><epage>e0242890</epage><pages>e0242890-e0242890</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33529230</pmid><doi>10.1371/journal.pone.0242890</doi><tpages>e0242890</tpages><orcidid>https://orcid.org/0000-0001-9483-2892</orcidid><orcidid>https://orcid.org/0000-0003-0064-4458</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2021-02, Vol.16 (2), p.e0242890-e0242890 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2485496449 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS) Journals Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Animals Antibodies Antigens Bioengineering Biology and life sciences Biotechnology Blood coagulation CHO Cells Cloning Clotting Coronaviridae Coronaviruses COVID-19 Cricetulus Deoxyribonucleic acid DNA Editing Electronic mail Epstein-Barr virus Follicle-stimulating hormone Gene Expression Genetic aspects Genetic Vectors Glycoproteins Glycosylation Humans Laboratories Long terminal repeat Mammals Medicine and health sciences Methodology Middle East respiratory syndrome Pandemics Peptide Elongation Factor 1 - genetics Plasmids Polymerase chain reaction Protein Domains Protein S Proteins Research and Analysis Methods Research facilities Respiration Respiratory diseases Serology Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Social research Spike Glycoprotein, Coronavirus - chemistry Spike Glycoprotein, Coronavirus - genetics Spike Glycoprotein, Coronavirus - isolation & purification Structure Transfection - methods Vectors (Biology) Viral diseases Viral proteins Virus research Viruses Yeasts |
| title | High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector |
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