Q sepharose ion exchange column

Recombinant wild-type and mutant proteins were expressed and purified as described previously [13 (link), 21 (link)]. In brief, wild-type protein and deletion mutants were overexpressed in E. coli BL21(DE3)pLysS cells (Invitrogen). The cells were lysed in 50 mM Tris-HCl, 2 mM EDTA, and 0.1M NaCl (pH 7.5) containing lysozyme (0.1 mg/ml) and treated with benzonase (Sigma) for removal of DNA. Both of the mutant proteins went into inclusion bodies. Therefore, we used 6 M urea to solubilize inclusion bodies and then precipitated the recombinant proteins with 35% ammonium sulfate. The pellet was then dissolved in cell lysis buffer and purified using ion-exchange chromatography. Wild-type αA-crystallin was initially purified using gel filtration chromatography (Superdex G200 column). The fraction containing wild-type protein was treated with 6 M urea and processed further, as described for mutants. A stepwise gradient of 0 to 1 M NaCl was used for elution of proteins from the ion-exchange column. Refolding and purification of the mutant protein was achieved by on-column refolding on a Q-Sepharose ion-exchange column (GE Biosciences) [22 (link)]. The fractions were subjected to SDS-PAGE, and fractions containing crystallins were dialyzed against 50 mM Tris-HCl, concentrated and stored in presence of 10 mM dithiothreitol at −80 °C for further use.

Purification of native AQP1 was carried out using, at a time, one unit of outdated human red blood cells (RBC; NZ blood bank). The procedure adopted followed that described in van Hoek et al. (54 (link)) with some modifications. Following solubilization of AQP1-enriched membranes produced by stripping of the ghost membranes with 3% N-lauryl-sarcosine (NLS), the solubilized material (in 400 mM βOG) was loaded onto a Q-Sepharose ion exchange column (GE Healthcare Life Sciences, Milwaukee, WI, USA) washed with up to 150 mM NaCl and eluted at 250 mM NaCl in Tris buffer (Tris-HCl pH 8.0, ~50 mM NaCl, 0.1 mM EDTA, 40 mM βOG). Fractions containing AQP1 were pooled and treated with recombinant PNGaseF produced in-house, as described in Loo et al. (55 ), for generating deglycosylated AQP1. The amount of PNGaseF used varied from one-tenth to half of the amount of AQP1 present (estimated by absorbance at 280 nm), depending on enzyme activity. AQP1 was incubated with PNGaseF for 48 to 72 h at 18 °C. PNGaseF was removed by a second Q Sepharose chromatography run, as described above but using a smaller resin volume to enable concentration of purified AQP1. AQP1 was concentrated further using a CENTRICON with 50 kD molecular weight cutoff and loaded onto a Superdex S200 10/300 GL (GE Healthcare Life Sciences) size-exclusion column as a final purification step.