recombinant spcas9 protein  (Intact Genomics)

Journal: Biomedicines

Article Title: Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

doi: 10.3390/biomedicines12061226

Figure Lengend Snippet: ( A ) Genetic map of pET-28b-Cas9-His obtained from Addgene. ( B ) Transcriptional unit of SpCas9-NLS-6His.

Article Snippet: The recombinant SpCas9-His protein was produced using the pET-28b-Cas9-His plasmid acquired from Addgene (cat. no. 47327).

Techniques:

Journal: Biomedicines

Article Title: Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

doi: 10.3390/biomedicines12061226

Figure Lengend Snippet: Evaluation of the effects of IPTG concentration and induction temperature on the expression of SpCas9-His protein in BL21(DE3)-pLysS strain of E. coli . ( A ) SDS-PAGE analysis of the expression of SpCas9-His protein at 0.5 mM IPTG at 18 °C overnight. ( B ) SDS-PAGE analysis of the expression of SpCas9-His protein at 30 °C but at different IPTG concentrations (lane 1—0.5 mM IPTG, lane 2—protein ladder, lane 3—1 mM IPTG conc); ( C ) Expression of SpCas9-His protein at different induction temperatures (18, 25, 30, and 37 °C).

Article Snippet: The recombinant SpCas9-His protein was produced using the pET-28b-Cas9-His plasmid acquired from Addgene (cat. no. 47327).

Techniques: Concentration Assay, Expressing, SDS Page

Journal: Biomedicines

Article Title: Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

doi: 10.3390/biomedicines12061226

Figure Lengend Snippet: Assessment of SpCas9-His protein expression in BL21(DE3)-pLysS, BL21(DE3)-Star, Rosetta2, and BL21(DE3) cells induced with 0.5 mM IPTG at 30 °C for 6 h in LB medium. Legend—1 and 4: Cell lysate before induction; 2 and 5: Cell lysate post-induction; 3—Protein marker.

Article Snippet: The recombinant SpCas9-His protein was produced using the pET-28b-Cas9-His plasmid acquired from Addgene (cat. no. 47327).

Techniques: Expressing, Marker

Journal: Biomedicines

Article Title: Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

doi: 10.3390/biomedicines12061226

Figure Lengend Snippet: Evaluation of SpCas9-His protein expression in BL21(DE3)-pLysS induced with 0.5 mM IPTG. ( A ) Protein purification profile of SpCas9-His using Ni-Sepharose affinity chromatography. 1: protein ladder; 2—fraction from the wash step; 3—first 5 mL of the fraction from the elution step. This is an important step as it gets rid of the lower contamination and 4—remaining fraction from the elution step. ( B ) SDS PAGE gel after buffer exchange to ensure pure protein. 1: protein ladder; 2—pure protein after buffer exchange. ( C ) Western blot analysis of SpCas9-His expression. Legend: SpCas9-His protein and pre-stained protein MW marker.

Article Snippet: The recombinant SpCas9-His protein was produced using the pET-28b-Cas9-His plasmid acquired from Addgene (cat. no. 47327).

Techniques: Expressing, Protein Purification, Affinity Chromatography, SDS Page, Buffer Exchange, Western Blot, Staining, Marker

Journal: Biomedicines

Article Title: Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

doi: 10.3390/biomedicines12061226

Figure Lengend Snippet: ( A ) In vitro cleavage assay. Only a complex of SpCas9 and gRNA could digest the plasmid DNA. “C” represents commercial Cas9, while “IH” represents in-house Cas9. The numbers 30 and 60 denote the ratio of gRNA:SpCas9:DNA. For instance, 30 corresponds to a ratio of 30:30:1 (nM), and 60 corresponds to a ratio of 60:60:1 (nM). ( B ) The densitometric analysis of in vitro digestion assay of the cleaved DNA (784 bp) band, as monitored by agarose gel electrophoresis.

Article Snippet: The recombinant SpCas9-His protein was produced using the pET-28b-Cas9-His plasmid acquired from Addgene (cat. no. 47327).

Techniques: In Vitro, Cleavage Assay, Plasmid Preparation, Agarose Gel Electrophoresis