Glutathione sepharose column chromatography

Transformed E. coli BL-21 cells containing the pGEX-4T clones were cultured in 200 ml Luria broth and treated with 0.1 mM IPTG for 3 h. Cells were then harvested, washed with phosphate-buffered saline, and lysed by sonication in BugBuster Master Mix (Merck). The cell lysates were centrifuged at 13,000 g for 10 min at 4 °C. Recombinant GST and GST-DNAJC2 proteins were purified from the supernatants using glutathione-Sepharose column chromatography (GE Healthcare) and the purified proteins were concentrated using an Amicon Ultra-15 centrifugal filter device (Merck) [44 (link)].

Recombinant proteins tagged with glutathione-S-transferase (GST) were constructed by recombining the insertion sequences of pBluescript into pGEX-4T (GE Healthcare Life Sciences, Pittsburgh, PA) vector plasmids. The pBluescript plasmids were digested using a combination of EcoRI and XhoI or SmaI and XhoI. The inserted DNA fragments were isolated using GeneElute™ Minus EtBr SPIN COLUMNS (Sigma-Aldrich, St. Louis, MO). The insertion sequences were ligated in frame to SmaI- and XhoI-digested pGEX-4T-1 or EcoRI- and XhoI-digested pGEX-4T-3 using Ligation Convenience Kits (Nippon Gene, Toyama, Japan). The ligation mixtures were used to transform ECOS™ competent E. coli JM-109 (Nippon Gene) and appropriate recombinants were confirmed by DNA sequencing. The constructed pGEX recombinants with the correct insertion in the right orientation were then used to transform competent E. coli BL21-RIL-codon-plus (Stratagene). The expression of the GST-fusion proteins was induced by treating the transformed E. coli with 0.1 mM isopropyl-β-D-thiogalactoside for 3 h. The GST recombinant proteins were purified by glutathione-Sepharose column chromatography according to the manufacturer’s instructions (GE Healthcare Life Sciences) and dialyzed against 1 mM Tris–HCl (pH 7.5) and 1 mM EDTA.

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 ].

Transformed E. coli BL-21 cells containing pGEX-4T clones were cultured in 200 mL of Luria broth and treated with 1 mM IPTG for 3 h. The IPTG-treated cells were then harvested, washed with phosphate-buffered saline (PBS), and lysed by sonication in BugBuster Master Mix (Novagen, San Diego, CA). Subsequently, cell lysates were centrifuged at 13,000×g for 10 min at 4°C. The GST fusion recombinant proteins recovered in the supernatant fraction were directly affinity purified by glutathione-Sepharose column chromatography (GE Healthcare Life Sciences), according to the manufacturer’s instructions, and the purified proteins were concentrated using Amicon Ultra-15 Centrifugal Filter Devices (Merck Millipore, Darmstadt, Germany). The precipitates containing recombinant proteins were dissolved in 8 M urea in TED buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol], followed by dialysis stepwise against 4 and 2 M urea in TED buffer for 1 h each. The samples were then dialyzed against TED buffer for more than 12 h and centrifuged at 10,000×g for 30 min at 4°C. The recombinant proteins recovered in the supernatant were purified using glutathione-Sepharose as previously described [16 , 17 (link), 25 (link), 28 (link), 42 , 43 (link)].

To generate the expression plasmids for the GST-fused JMJD6 protein, we integrated the JMJD6 cDNA sequence between 1075 and 1834 into the EcoRI/XhoI site of pGEX-4T-1 vector (Cytiva, Marlborough, MA, USA), as previously described [17 (link)].
Moreover, E. coli BL-21 cells transformed with the pGEX-4T-1 clone were cultured in 200 mL of Luria–Bertani broth and treated with 0.1 mM IPTG for 3 h. The cells were then collected and lysed by sonication in BugBuster Master Mix (Novagen, San Diego, CA, USA), followed by centrifugation at 13,000× g and 4 °C for 10 min. GST-tagged JMJD6 proteins were purified through Glutathione-Sepharose column chromatography (GE Healthcare Life Sciences, Chicago, IL, USA) and dialyzed as previously described [17 (link),18 (link),19 (link)].

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.