Recombinant human galectin-3 and mutants and Xenopus galectin-3 were produced in E. coli BL21Star (DE3) cells (Invitrogen) and purified by affinity chromatography on lactosyl-Sepharose essentially as described for wild type human galectin-3 (34 (link)) but with some variation to optimize yield for each protein. The initial yields ranged from 3 mg/liter culture for G182A to 80 mg/liter for R144S, but, for example, lowering the temperature of the isopropyl 1-thio-β-d -galactopyranoside induction from 37 to 30 °C increased the yield of G182A galectin-3 about 10-fold. Mouse galectin-3 was produced from vector pIN III ompA2 in E. coli JA221 cells as previously described (36 (link)), except that Tryptone soy broth was used. The bacteria were processed and galectin-purified in the same way as for human galectin-3 described above. The galectins, in phosphate-buffered saline (118 mm NaCl, 67 mm Na+/K+-phosphate, pH 7.2) containing 4 mm β-mercaptoethanol, 2 mm EDTA and 150 mm lactose, were dialyzed against 2 liters of water that was changed once every 2 h 7 times and lyophilized and stored at −20 °C until use. This procedure does not completely deplete the galectin-bound lactose, which helps to preserve stability. Before use, the galectins were dissolved in the appropriate buffer, and any remaining lactose was removed by repeated ultrafiltration and concentration in a Centricon Plus-70, Ultracel PL10, or Centriprep Y10 ultrafiltration cell (Millipore AB, Sundbyberg, Sweden). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Sundbyberg, Sweden). Protein size and integrity was determined by SDS-PAGE using 4–20% PreciseTM Protein Gels from Pierce (Nordic Biolabs, Täby, Sweden) in Tris/HEPES running buffer.
Differential scanning calorimetry measurements were performed on a MicroCal differential scanning calorimeter (MicroCal Inc., Northampton, MA) with a cell volume of 0.5072 ml. All samples were degassed for 15 min at room temperature before scanning. Protein samples (0.5 mg/ml) in PBS buffer with or without ligand were scanned in the temperature range 25 to 80 °C at a rate of 1 °C/min.The reversibility of the calorimetric traces was assessed by the reproducibility of scans upon rapid cooling to 25 °C followed by rescanning. Base-line scans were collected with buffer in both the reference and sample cells.
Differential scanning calorimetry measurements were performed on a MicroCal differential scanning calorimeter (MicroCal Inc., Northampton, MA) with a cell volume of 0.5072 ml. All samples were degassed for 15 min at room temperature before scanning. Protein samples (0.5 mg/ml) in PBS buffer with or without ligand were scanned in the temperature range 25 to 80 °C at a rate of 1 °C/min.The reversibility of the calorimetric traces was assessed by the reproducibility of scans upon rapid cooling to 25 °C followed by rescanning. Base-line scans were collected with buffer in both the reference and sample cells.
