Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4 °C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol.
Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4°C in 10 mM Tris-HCl, 500 mM NaCl, and 5% glycerol.
Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4°C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol.
recombinant enzyme from aroE auxotrophic mutant strain AB2834 by ammonium sulfate fractionation, anion exchange chromatography, ultrafiltration, and gel filtration
recombinant GST-tagged enzyme from Escherichia coli strain BL21-CodonPlus(DE3)-RIL by glutathione affinity chromatography and tag cleavage through thrombin
recombinant GST-tagged SeMet-labeled enzyme from Escherichia coli strain BL21 by glutathione affinity and ion exchange chromatography, proteolytic removal of the GST-tag
recombinant soluble enzyme 8.5fold from Escherichia coli strain BL21(DE3) by ion exchange and hydrophobic interaction chromatography, gel filtration, and again ion exchange chromatography, to homogeneity