A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications
Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant <i>Streptococcus pyogenes</i> Cas9 (<i>Sp</i>Cas9) at some point during their application, for instance, for in vi...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-05-01
|
| Series: | Methods and Protocols |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2409-9279/5/3/44 |
| _version_ | 1797483866018021376 |
|---|---|
| author | Andrea Luciana Fleitas Mario Señorale Sabina Vidal |
| author_facet | Andrea Luciana Fleitas Mario Señorale Sabina Vidal |
| author_sort | Andrea Luciana Fleitas |
| collection | DOAJ |
| description | Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant <i>Streptococcus pyogenes</i> Cas9 (<i>Sp</i>Cas9) 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 <i>Sp</i>Cas9 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−<i>Sp</i>Cas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%. |
| first_indexed | 2024-03-09T22:54:11Z |
| format | Article |
| id | doaj.art-67c943b0b7cf48bd98f2e9f8cb7b2d38 |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-09T22:54:11Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj.art-67c943b0b7cf48bd98f2e9f8cb7b2d382023-11-23T18:14:42ZengMDPI AGMethods and Protocols2409-92792022-05-01534410.3390/mps5030044A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro ApplicationsAndrea Luciana Fleitas0Mario Señorale1Sabina Vidal2Laboratorio de Biología Molecular Vegetal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo 11400, UruguaySección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Montevideo 11400, UruguayLaboratorio de Biología Molecular Vegetal, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo 11400, UruguayGenome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant <i>Streptococcus pyogenes</i> Cas9 (<i>Sp</i>Cas9) 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 <i>Sp</i>Cas9 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−<i>Sp</i>Cas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.https://www.mdpi.com/2409-9279/5/3/44CIRSPR/Cas9expressionpurificationimmobilized metal affinity chromatographyion exchange |
| spellingShingle | Andrea Luciana Fleitas Mario Señorale Sabina Vidal A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications Methods and Protocols CIRSPR/Cas9 expression purification immobilized metal affinity chromatography ion exchange |
| title | A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications |
| title_full | A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications |
| title_fullStr | A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications |
| title_full_unstemmed | A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications |
| title_short | A Robust Expression and Purification Method for Production of <i>Sp</i>Cas9-GFP-MBP Fusion Protein for In Vitro Applications |
| title_sort | robust expression and purification method for production of i sp i cas9 gfp mbp fusion protein for in vitro applications |
| topic | CIRSPR/Cas9 expression purification immobilized metal affinity chromatography ion exchange |
| url | https://www.mdpi.com/2409-9279/5/3/44 |
| work_keys_str_mv | AT andrealucianafleitas arobustexpressionandpurificationmethodforproductionofispicas9gfpmbpfusionproteinforinvitroapplications AT mariosenorale arobustexpressionandpurificationmethodforproductionofispicas9gfpmbpfusionproteinforinvitroapplications AT sabinavidal arobustexpressionandpurificationmethodforproductionofispicas9gfpmbpfusionproteinforinvitroapplications AT andrealucianafleitas robustexpressionandpurificationmethodforproductionofispicas9gfpmbpfusionproteinforinvitroapplications AT mariosenorale robustexpressionandpurificationmethodforproductionofispicas9gfpmbpfusionproteinforinvitroapplications AT sabinavidal robustexpressionandpurificationmethodforproductionofispicas9gfpmbpfusionproteinforinvitroapplications |