Ngctm chromatography system

Each Ec and Hb was analyzed using a NGC^TM Chromatography System (BioRad, Hercules, CA) with a BioRad Enrich^TM SEC 650 10 x 300 column with a total volume of 24 ml (Cat. #780–1650). Samples were eluted from the column using 20 mM Tris buffer, pH 7.4 at a flow rate of 0.5 ml/min. Distinct peak fractions were detected via absorbance at 280 nm and collected separately for further analysis (e.g., thermal stability and PAGE). The elution profiles shown in Figure 2 were normalized such that the maxima of the first elution peak were held constant.

A scale-up culture (0.5 L) was conducted for the purification of recombinant proteins using Ni-affinity chromatography. The harvested cells were suspended in 10 mL B-PER and sonicated as previously described [24 (link)], or mildly lysed in 10 mL B-PER, and then the two different methods were compared. Supernatants were obtained by centrifugation at 15,000× g for 12 min, and were loaded on to a nickel column equilibrated with A buffer [50 mL Tris-Cl (pH 7.5), 300 mM NaCl, 10% glycerol, 10 mM imidazole, 2 mM beta-mercaptoethanol, and 0.1% Tween-20]. After washing with the A buffer, target proteins were eluted with a concentration gradient of imidazole. Each fraction was analyzed by SDS-PAGE, and the fractions enriched with the target protein were pooled and dialyzed against the storage buffer [50 mM Tris-Cl (pH 7.5) and 300 mM NaCl].
For SEC, some of the fractions from the Ni-affinity chromatography were loaded on to a Superdex 200 Increase 10/300 GL column or a 10/300 Superose^TM 6 Increase column (GE Healthcare, Chicago, IL, USA). Before loading, the gel filtration column was equilibrated with two column volumes of buffer [50 mM Tris-Cl (pH 7.5) and 300 mM NaCl]. Proteins were loaded at 3% column volume, and eluted at a flow rate of 0.3 mL/min using a NGC^TM chromatography system (Bio-Rad Laboratories, Irvine, CA, USA).

For protein expression, overnight cultures of E. coli containing plasmids of either DisA or YybT were inoculated into 1 L LB medium and cultured at 37 °C. At OD₆₀₀ of 0.6, the cultures were supplemented with 1 mM IPTG to induce expression and incubated at 16 °C with 250 rpm for 18 h. The induced cells were centrifuged at 4 °C and pellets resuspended in lysis buffer [50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl for DisA and 10 mM Tris-HCl, pH 8.0, 100 mM NaCl for YybT]. The resuspended cells were lysed by sonication and centrifuged at 25,000 rpm for 25 min at 4 °C. The supernatants were passed through HisTrap HP 1 mL columns (GE) and the proteins purified using the Bio-Rad NGC^TM Chromatography System at a 1 mL/min flowrate. Elution of proteins was achieved by adding 200 mM imidazole to the lysis buffer. The concentrations of the purified proteins were determined by measuring their A₂₈₀.