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A Robust Expression and Purification Method for Production of SpCas9-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 Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (S...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9228339/ https://www.ncbi.nlm.nih.gov/pubmed/35736545 http://dx.doi.org/10.3390/mps5030044 |
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author | Fleitas, Andrea Luciana Señorale, Mario Vidal, Sabina |
author_facet | Fleitas, Andrea Luciana Señorale, Mario Vidal, Sabina |
author_sort | Fleitas, Andrea Luciana |
collection | PubMed |
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%. |
format | Online Article Text |
id | pubmed-9228339 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-92283392022-06-25 A Robust Expression and Purification Method for Production of SpCas9-GFP-MBP Fusion Protein for In Vitro Applications Fleitas, Andrea Luciana Señorale, Mario Vidal, Sabina Methods Protoc Protocol 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%. MDPI 2022-05-28 /pmc/articles/PMC9228339/ /pubmed/35736545 http://dx.doi.org/10.3390/mps5030044 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Protocol Fleitas, Andrea Luciana Señorale, Mario Vidal, Sabina A Robust Expression and Purification Method for Production of SpCas9-GFP-MBP Fusion Protein for In Vitro Applications |
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 | robust expression and purification method for production of spcas9-gfp-mbp fusion protein for in vitro applications |
topic | Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9228339/ https://www.ncbi.nlm.nih.gov/pubmed/35736545 http://dx.doi.org/10.3390/mps5030044 |
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