Hiload 16 600 superdex 75 pg gel filtration column

The ADC-7 β-lactamase was purified from Escherichia coli BL21(DE3) cells carrying the pET-24a(+)bla_ADC-7 plasmid. Cells were grown in super optimal broth (SOB) at 37°C to an approximate optical density at λ 600 nm (OD₆₀₀) of 0.6 to 0.8 and induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for a minimum of 3 h to express the β-lactamase. The cells were pelleted and frozen at −20°C for ≥12 h prior to lysis in 50 mM Tris-HCl buffer, pH 7.4, containing 40 mg/ml lysozyme, 0.1 mM magnesium sulfate, and 250 U benzonase nuclease. The supernatant was further purified by preparative isoelectric focusing (pIEF), eluted from the Sephadex 10 mM phosphate-buffered saline (PBS) at pH 7.4, and polishing steps were completed on a HiLoad 16/600 Superdex 75 pg gel filtration column (GE Healthcare). The final sample of protein was concentrated using centrifugal filter units with a molecular weight cutoff of 10,000 daltons (Millipore). The purity of the protein was assessed by quadrupole time of flight (Q-TOF) mass spectrometry and Coomassie blue-stained SDS-PAGE.