To construct the glutathione-S-transferase (GST)-fused protein expression plasmids, the cDNA sequences were recombined into the pGEX-4T vector (GE Healthcare Life Sciences, Pittsburgh, PA, USA) as previously described (32 (link), 35 (link), 37 (link)). The pBluescript plasmids associated with the cDNA inserts were digested with the restriction endonucleases EcoRI and XhoI and detached by agarose gel electrophoresis. GenElute Minus EtBr spin columns (Sigma-Aldrich Corp., St. Louis, MO, USA) were used to isolate the cDNA fragments which were ligated in frame to EcoRI- and XhoI-digested pGEX-4T-3 linearized vectors with ligation convenience kits (Nippon Gene, Toyama, Japan). The ligation mixtures were used to transform ECOS-competent E. coli BL-21 cells (Nippon Gene). Successful recombination was confirmed by DNA sequencing and protein expression analysis.
Pgex 4t 3
Manufactured by Nippon Gene
Sourced in Japan
The PGEX-4T-3 is a plasmid vector used for the expression of recombinant proteins in Escherichia coli. It contains the tac promoter, which is inducible by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG), and a glutathione S-transferase (GST) tag for purification of the expressed protein.
Automatically generated - may contain errors
Lab products found in correlation
Ligation convenience kit, by Nippon Gene (5 mentions)
Pgex 4t 3, by GE Healthcare (4 mentions)
Glutathione sepharose column chromatography, by GE Healthcare (4 mentions)
Genelute minus etbr spin columns, by Merck Group (1 mentions)
E coli bl 21 cells, by Nippon Gene (1 mentions)
Pgex 4t vector, by GE Healthcare (1 mentions)
Ecos competent e coli bl 21, by Nippon Gene (1 mentions)
5 protocols using pgex 4t 3
1
Construction of GST-Fused Protein Plasmids
2
Constructing and Purifying GST-Fused Recombinant Proteins
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We constructed the expression plasmids of GST‐fused proteins by recombining the cDNA sequences into pGEX‐4T‐3 (GE Healthcare Life Sciences). Next, the inserted DNA fragments were ligated into pGEX‐4T‐3 using Ligation Convenience Kits (Nippon Gene). We used ligation mixtures to transform ECOS™‐competent E. coli BL21 (DE3; Nippon Gene) and confirmed appropriate recombinants by DNA sequencing and protein expression analyses. Next, the expression of the GST‐fusion proteins was induced by treating the transformed E. coli with 0.1 mmol/L IPTG for 3 hours. We purified the GST‐fused recombinant proteins by glutathione sepharose column chromatography as per the manufacturer's instructions (GE Healthcare Life Sciences) and dialyzed against phosphate‐buffered saline, as described previously.29
3
Purification of GST-Fusion Proteins
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The expression plasmids of glutathione S- transferase (GST)-fused proteins were constructed by recombining the cDNA sequences into pGEX-4T-3 (GE Healthcare Life Sciences, Pittsburgh, PA). The inserted DNA fragments were ligated in frame to pGEX-4T-3 using the Ligation Convenience Kits (Nippon Gene, Toyama, Japan). Ligation mixtures were used to transform ECOS™-competent E. coli BL-21 (Nippon Gene), and appropriate recombinants were confirmed by DNA sequencing as well as protein expressions. Treating the transformed E. coli with 0.1 mM IPTG for 3 h induced the expression of the GST-fusion proteins. The GST-fused recombinant proteins were purified by Glutathione Sepharose column chromatography according to the manufacturer's instructions (GE Healthcare Life Sciences) and dialyzed against phosphate-buffered saline as described in previous studies [20 –22 ].
4
Recombinant Protein Expression and Purification
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We constructed the expression plasmids of glutathione‐S‐transferase (GST)‐fused proteins by recombining the cDNA sequences into pGEX‐4 T‐3 (GE Healthcare Life Sciences). Next, the inserted DNA fragments were ligated into pGEX‐4 T‐3 using Ligation Convenience Kits (Nippon Gene). We used ligation mixtures to transform ECOS™‐competent E. coli BL21 (DE3; Nippon Gene) and confirmed appropriate recombinants using DNA sequencing and protein expression analyses. The expression of the GST‐fusion proteins was then induced by treating the transformed E. coli with 0.1 mM IPTG for 3 h. We purified GST‐fused recombinant proteins by Glutathione‐Sepharose column chromatography per the manufacturer's instructions (GE Healthcare Life Sciences) and dialyzed against phosphate‐buffered saline, as previously described.
34 Confirmation of purification has been described in previous studies.
34 Confirmation of purification has been described in previous studies.
5
Recombinant GST-Fusion Protein Expression and Purification
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The expression plasmids of GST‐fused proteins were constructed by recombining the cDNA sequences into pGEX‐4T‐3 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The inserted DNA fragments were ligated into pGEX‐4T‐3 using Ligation Convenience Kits (Nippon Gene). Ligation mixtures were used for transforming ECOS‐competent E. coli BL21 (DE3) (Nippon Gene), and appropriate recombinants were confirmed by DNA sequencing as well as protein expression analyses. Treating the transformed E. coli with 0.1 mmol/L IPTG for 3 hours induced the expression of the GST‐fusion proteins. The GST‐fused recombinant proteins were purified by glutathione sepharose column chromatography in accordance with the manufacturer's instructions (GE Healthcare Life Sciences) and dialyzed against PBS as described in previous studies.36 , 37
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