His trap hp ni nta columns

The mrx1 gene (cg0964) was amplified from chromosomal DNA of C. glutamicum ATCC13032 by PCR using the primer pair Cgmrx1-roGFP2-NdeI-FOR and pQE60-Cgmrx1-roGFP2-SpeI-REV. The PCR product was digested with NdeI and SpeI and cloned into plasmid pET11b-brx-roGFP2[42] (link) to exchange the brx sequence by mrx1 with generation of plasmid pET11b-mrx1-roGFP2(Table S1). The correct sequence was confirmed by PCR and DNA sequencing.
The E. coli BL21 (DE3) plysS expression strain containing the plasmid pET11b-mrx1-roGFP2 was grown in 1 l LB medium until OD₆₀₀ of 0.6 at 37 °C, followed by induction with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 16 h at 25 °C. Recombinant His₆-tagged Mrx1-roGFP2 protein was purified using His Trap™ HP Ni-NTA columns (5 ml; GE Healthcare, Chalfont St Giles, UK) and the ÄKTA purifier liquid chromatography system (GE Healthcare) according to the instructions of the manufacturer (USB). The purified protein was dialyzed against 10 mM Tris-HCl (pH 8.0), 100 mM NaCl and 30% glycerol and stored at − 80 °C. Purity of the protein was analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue (CBB) staining.

The DIP1310 gene encoding GapDH was amplified by PCR using the primer pairs Gap-for (5′-GGAATTCCATATGGTGACGATTCGCGTAGGTATCA-3′) and Gap-rev (5′-CTAGCTAGCTTAGTGATGGTGATGGTGATGGAGACGCTCACCGACGTATTC-3′) with C. diphtheriae DSM43989 chromosomal DNA as template. The PCR product was digested with NheI and NdeI restriction enzymes and cloned into a similarly digested pET11b expression vector resulting in pET11b-gapDH that was transformed into E. coli BL21(DE3)plysS. The gapDH sequence was confirmed by DNA sequencing. For GapDH overproduction, the E. coli BL21(DE3)plysS strain with plasmid pET11b-gap was cultured in LB broth medium to an OD₆₀₀ of 0.5 to 0.7 at 37 °C. Protein expression was induced with 1 mM IPTG (Isopropyl-β-D-1-thiogalactopyranoside) and cultivation was continued for 4 hours. Recombinant His₆-tagged GapDH was purified by affinity chromatography using His Trap™ HP Ni-NTA columns (5 ml; GE Healthcare, Chalfont St Giles, UK) and the ÄKTA purifier liquid chromatography system (GE Healthcare) according to the instructions of the manufacturers. Purified GapDH was dialyzed against 20 mM Tris-HCl, pH 8.0 and concentrated to 20 mg/ml using Vivaspin Ultra concentrators (Sartorius, Göttingen, Germany). The cloning and purifications of recombinant His6-tagged proteins Mrx1, Mtr, Trx and TrxR were performed as described previously^{59 (link)}.

The recombinant His-tagged PPARγ was expressed as previously described [21 (link)]. Briefly, the plasmids (pET10, pET11, pET12, pET13, pET14, or pET15) were transformed to Escherichia coli BL-21 cells and seeded on Luria-Bertani medium added with 50 μg/ml kanamycin (Solarbio, China) at 37°C. When the OD₆₀₀ reached approximately 0.6, 0.5 mM isopropyl-β-D-thiogalactopyranoside (Solarbio, China) was added to induce the protein overexpression and then shaken-incubated at 22°C for 18 h. After harvesting by centrifugation at 4°C, 5500 rpm for 10 min, cells were resuspended in lysis buffer (500 mM NaCl, 50 mM HEPES, 5 mM imidazole, and 5% [v/v] glycerol; pH = 7.5) and disrupted by an ultrasonicator for 30 min. And the lysate was centrifugated at 4°C, 15,000 rpm for 30 min. After filtering through a 0.45 μm cellulose acetate syringe filter, the supernatant containing the target protein was loaded onto HisTrap™ HP Ni-NTA columns (LOT 10236775, GE, USA). The recombinant His-tagged PPARγ was purified following the instructions.