Hitrap heparin hp 5ml column

Proteins were expressed as His₆ tag or His₆SUMO3 fusion proteins in Escherichia coli BL21(DE3) cells grown to an OD₆₀₀ of 0.6 before the temperature was reduced to 22°C and protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM. Cells were lysed and the fusion proteins purified from the soluble fraction of lysate by nickel-affinity chromatography. HRV 3C protease was used to cleave the His₆ tag or His₆SUMO3 tag by incubation overnight at 4°C then both the tags and protease were separated from the cleaved protein by nickel-affinity chromatography. The protein was purified further on a HiTrap Heparin HP 5ml column (GE Healthcare) to remove nucleic acid contamination and a final purification step of size exclusion chromatography was performed using an ÄKTA purifier system (GE Healthcare) with a Hiload S75 16/60 Superdex prep grade column (GE Healthcare).
Fractions of pure protein were pooled and concentrated before being dialyzed into a final buffer of 10 mM phosphate pH 6.9, 50 mM NaCl, 1 mM TCEP, 10 μM ZnCl₂ and concentrated in a Vivaspin MWCO 10 000. Protein concentration was determined from the absorbance at 280 nm.

The E. coli BL21(DE3) cells carrying pET-28a-σ^A were cultured at 37 °C in LB medium to OD₆₀₀ of 0.6–0.8. Protein expression was induced by 0.5 mM IPTG at 18 °C for 14 h. Cells were harvested and pellets were suspended in lysis buffer B (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 5% (v/v) glycerol, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Bimake.com Inc.)) and lysed using an Avestin EmulsiFlex-C3 cell disrupter (Avestin Inc.). The supernatant of the lysate was loaded onto a gravity column packed with 2 ml Ni-NTA agarose (Smart Lifesciences Inc., China), which was washed with lysis buffer B containing 20 mM imidazole and eluted with lysis buffer B containing 300 mM imidazole. The eluted fractions were dialyzed overnight in the dialysis buffer as described above. The sample were loaded onto a Heparin column (HiTrap Heparin HP 5 ml column, GE Healthcare Life Sciences), and eluted with a salt gradient of buffer A (50 mM Tris-HCl, pH 8.0, 0.05 M NaCl, 5% (v/v) glycerol, 1 mM DTT) and buffer B (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 5% (v/v) glycerol, 1 mM DTT). The elute fractions containing Bs σ^A were concentrated to ~4 mg/ml and stored at −80 °C.

The expression of Bs-BmrR was induced by 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 18 °C for 14 h in E. coli BL21(DE3) cells carrying pTolo-EX5-Bs-BmrR. The cells were lysed in lysis buffer A (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 5% (v/v) glycerol, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Bimake.com Inc.)) using an Avestin EmulsiFlex-C3 cell disrupter (Avestin Inc.). The Bs-BmrR were enriched by a gravity column packed with 2 ml Ni-NTA agarose (Smart Life Sciences Inc.), washed with lysis buffer A containing 20 mM imidazole, and eluted with lysis buffer A containing 300 mM imidazole. The eluted fractions were treated with TEV protease and dialyzed overnight in a dialysis buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5% (v/v) glycerol, 5 mM β-mercaptoethanol). The sample was reloaded onto a Ni-NTA column to remove the impurity. The fraction containing Bs-BmrR were further loaded onto a Heparin column (HiTrap Heparin HP 5 ml column, GE Healthcare Life Sciences), and eluted with a salt gradient of buffer A (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT)) and buffer B (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 5% (v/v) glycerol, 1 mM DTT). The elute fractions containing target proteins were collected, concentrated to 2 mg/ml, and stored at −80 °C.