A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications
Resumen:
Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.
| 2022 | |
| ANII: FMV_3_2018_1_148011 | |
|
CIRSPR/Cas Expression Purification Immobilized metal affinity chromatography Ion exchange |
|
| Inglés | |
| Universidad de la República | |
| COLIBRI | |
| https://hdl.handle.net/20.500.12008/41377 | |
| Acceso abierto | |
| Licencia Creative Commons Atribución (CC - By 4.0) |
| _version_ | 1807522802593431552 |
|---|---|
| author | Fleitas, Andrea Luciana |
| author2 | Señorale, Mario Vidal, Sabina |
| author2_role | author author |
| author_facet | Fleitas, Andrea Luciana Señorale, Mario Vidal, Sabina |
| author_role | author |
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| bitstream.checksumAlgorithm.fl_str_mv | MD5 MD5 MD5 MD5 MD5 |
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| collection | COLIBRI |
| dc.contributor.filiacion.none.fl_str_mv | Fleitas Andrea Luciana, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica. Señorale Mario, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología. Vidal Sabina, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica. |
| dc.creator.none.fl_str_mv | Fleitas, Andrea Luciana Señorale, Mario Vidal, Sabina |
| dc.date.accessioned.none.fl_str_mv | 2023-11-21T14:36:18Z |
| dc.date.available.none.fl_str_mv | 2023-11-21T14:36:18Z |
| dc.date.issued.none.fl_str_mv | 2022 |
| dc.description.abstract.none.fl_txt_mv | Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%. |
| dc.description.sponsorship.none.fl_txt_mv | ANII: FMV_3_2018_1_148011 |
| dc.format.extent.es.fl_str_mv | 10 h. |
| dc.format.mimetype.es.fl_str_mv | application/pdf |
| dc.identifier.citation.es.fl_str_mv | Fleitas, A, Señorale, M y Vidal, S. "A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications". Methods and Protocols. [en línea] 2022, 5(3): 44. 10 h. DOI: 10.3390/mps5030044 |
| dc.identifier.doi.none.fl_str_mv | 10.3390/mps5030044 |
| dc.identifier.issn.none.fl_str_mv | 2409-9279 |
| dc.identifier.uri.none.fl_str_mv | https://hdl.handle.net/20.500.12008/41377 |
| dc.language.iso.none.fl_str_mv | en_US eng |
| dc.publisher.es.fl_str_mv | MDPI |
| dc.relation.ispartof.es.fl_str_mv | Methods and Protocols, 2022, 5(3): 44. |
| dc.rights.license.none.fl_str_mv | Licencia Creative Commons Atribución (CC - By 4.0) |
| dc.rights.none.fl_str_mv | info:eu-repo/semantics/openAccess |
| dc.source.none.fl_str_mv | reponame:COLIBRI instname:Universidad de la República instacron:Universidad de la República |
| dc.subject.es.fl_str_mv | CIRSPR/Cas Expression Purification Immobilized metal affinity chromatography Ion exchange |
| dc.title.none.fl_str_mv | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| dc.type.es.fl_str_mv | Artículo |
| dc.type.none.fl_str_mv | info:eu-repo/semantics/article |
| dc.type.version.none.fl_str_mv | info:eu-repo/semantics/publishedVersion |
| description | Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | COLIBRI_af1053c3aec5cb56dd79377d95f807b4 |
| identifier_str_mv | Fleitas, A, Señorale, M y Vidal, S. "A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications". Methods and Protocols. [en línea] 2022, 5(3): 44. 10 h. DOI: 10.3390/mps5030044 2409-9279 10.3390/mps5030044 |
| instacron_str | Universidad de la República |
| institution | Universidad de la República |
| instname_str | Universidad de la República |
| language | eng |
| language_invalid_str_mv | en_US |
| network_acronym_str | COLIBRI |
| network_name_str | COLIBRI |
| oai_identifier_str | oai:colibri.udelar.edu.uy:20.500.12008/41377 |
| publishDate | 2022 |
| reponame_str | COLIBRI |
| repository.mail.fl_str_mv | mabel.seroubian@seciu.edu.uy |
| repository.name.fl_str_mv | COLIBRI - Universidad de la República |
| repository_id_str | 4771 |
| rights_invalid_str_mv | Licencia Creative Commons Atribución (CC - By 4.0) |
| spelling | Fleitas Andrea Luciana, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica.Señorale Mario, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología.Vidal Sabina, Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Química Biológica.2023-11-21T14:36:18Z2023-11-21T14:36:18Z2022Fleitas, A, Señorale, M y Vidal, S. "A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications". Methods and Protocols. [en línea] 2022, 5(3): 44. 10 h. DOI: 10.3390/mps50300442409-9279https://hdl.handle.net/20.500.12008/4137710.3390/mps5030044Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.Submitted by Farías Verónica (vfarias@fcien.edu.uy) on 2023-11-21T13:28:53Z No. of bitstreams: 2 license_rdf: 24251 bytes, checksum: 71ed42ef0a0b648670f707320be37b90 (MD5) 103390mps5030044.pdf: 2602384 bytes, checksum: 1914d2f726147f6c05fc91f14251b0de (MD5)Approved for entry into archive by Faget Cecilia (lfaget@fcien.edu.uy) on 2023-11-21T14:03:33Z (GMT) No. of bitstreams: 2 license_rdf: 24251 bytes, checksum: 71ed42ef0a0b648670f707320be37b90 (MD5) 103390mps5030044.pdf: 2602384 bytes, checksum: 1914d2f726147f6c05fc91f14251b0de (MD5)Made available in DSpace by Luna Fabiana (fabiana.luna@seciu.edu.uy) on 2023-11-21T14:36:18Z (GMT). No. of bitstreams: 2 license_rdf: 24251 bytes, checksum: 71ed42ef0a0b648670f707320be37b90 (MD5) 103390mps5030044.pdf: 2602384 bytes, checksum: 1914d2f726147f6c05fc91f14251b0de (MD5) Previous issue date: 2022ANII: FMV_3_2018_1_14801110 h.application/pdfen_USengMDPIMethods and Protocols, 2022, 5(3): 44.Las obras depositadas en el Repositorio se rigen por la Ordenanza de los Derechos de la Propiedad Intelectual de la Universidad de la República.(Res. Nº 91 de C.D.C. de 8/III/1994 – D.O. 7/IV/1994) y por la Ordenanza del Repositorio Abierto de la Universidad de la República (Res. Nº 16 de C.D.C. de 07/10/2014)info:eu-repo/semantics/openAccessLicencia Creative Commons Atribución (CC - By 4.0)CIRSPR/CasExpressionPurificationImmobilized metal affinity chromatographyIon exchangeA robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applicationsArtículoinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionreponame:COLIBRIinstname:Universidad de la Repúblicainstacron:Universidad de la RepúblicaFleitas, Andrea LucianaSeñorale, MarioVidal, SabinaLICENSElicense.txtlicense.txttext/plain; charset=utf-84267http://localhost:8080/xmlui/bitstream/20.500.12008/41377/5/license.txt6429389a7df7277b72b7924fdc7d47a9MD55CC-LICENSElicense_urllicense_urltext/plain; charset=utf-844http://localhost:8080/xmlui/bitstream/20.500.12008/41377/2/license_urla0ebbeafb9d2ec7cbb19d7137ebc392cMD52license_textlicense_texttext/html; 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- Universidad de la Repúblicafalse |
| spellingShingle | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications Fleitas, Andrea Luciana CIRSPR/Cas Expression Purification Immobilized metal affinity chromatography Ion exchange |
| status_str | publishedVersion |
| title | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| title_full | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| title_fullStr | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| title_full_unstemmed | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| title_short | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| title_sort | A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications |
| topic | CIRSPR/Cas Expression Purification Immobilized metal affinity chromatography Ion exchange |
| url | https://hdl.handle.net/20.500.12008/41377 |
