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Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.12008/41377 How to cite
Title: A robust expression and purification method for production of SpCas9-GFP-MBP fusion protein for In vitro applications
Authors: Fleitas, Andrea Luciana
Señorale, Mario
Vidal, Sabina
Type: Artículo
Keywords: CIRSPR/Cas, Expression, Purification, Immobilized metal affinity chromatography, Ion exchange
Issue Date: 2022
Abstract: 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%.
Publisher: MDPI
IN: Methods and Protocols, 2022, 5(3): 44.
Sponsors: ANII: FMV_3_2018_1_148011
Citation: 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
ISSN: 2409-9279
Appears in Collections:Publicaciones académicas y científicas - Facultad de Ciencias

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