α lactose agarose resin

The protein expression and purification are described elsewhere [50 (link),51 (link)]. Briefly, proteins’ sequence in pET11a vector was transformed in BL21(DE3) E. coli. For labelled protein, a 5 mL overnight culture was added in one liter of M9 media containing antibiotic and ¹⁵N-NH₄Cl (1 g) as the nitrogen source. When the culture reached 0.7–1.2 of OD₆₀₀, protein expression was induced by the addition of 1 mM Isopropyl β-D-1-thio-galactopyranoside (IPTG) and growth continued for 3 h at 37 °C. After, cells were harvested and resuspended in a column buffer (PBS 1× pH 7.2, 2 mM EDTA, 2 mM β-mercarptoethanol /DTT, 0.1% NaN3) and 1 mM PMSF was added to inhibit proteases cleavage. The suspension was sonicated and the crude extract clarified by centrifugation at 35,000× g rpm for 30 min at 4 °C. The soluble fraction was loaded onto a pre-equilibrated α-Lactose-Agarose resin (Sigma-Aldrich) column and washed with 50 mL of column buffer. To elute recombinant h-Gal8N-ter 7 mL of elution buffer (150 mM α-Lactose in column buffer) was injected. Protein purity was checked by SDS-PAGE and subsequently confirmed by LC-MS.
Both galectins were thoroughly dialyzed against PBS, pH 7.4 until no lactose was present, before use.

The purification was done by following the protocol described by Rajan et al. [13 (link)] with minor modifications. Approximately 5 mL of α-lactose agarose resin (Sigma-Aldrich, St. Louis, MO, USA) in a 50 mL tube was washed twice with 3 volumes of 15 MΩ/cm analytic grade water. Washed resin was equilibrated with 3 volumes of binding buffer at 4 °C, and 20 mL of prepared skin mucus supernatant was added and allowed to bind for 2 h at 4 °C under rotation, followed by three times washing with binding buffer. The resin was loaded to a 10 ml Bio-Rad gravity purification column. The lactose binding protein was eluted manually using elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.5 M α-lactose).
Ten fractions of eluate, 500 µL each, were collected and the protein concentration in each fraction quantified by Qubit® Protein Assay Kit in a Qubit® fluorometer (Life Technologies, Camarillo, CA, USA). Each fraction containing protein was then desalted.