Sp sepharose hp

The solvent was loaded onto a 55 mL SP Sepharose HP (2.6 × 10 cm, 26/10, GE Healthcare, Little Chalfont, England) equilibrated with a 5 L bed volume of running buffer (50 mM potassium acetate, pH 4.7, and 1% n-octyl-β-d-glucoside (w/v)). Bound proteins were eluted from the column using a NaCl gradient (from 0 to 1000 mM). Active fractions were concentrated using a Centricon 100 (Amicon, Danvers, MA, USA) at 4700× g to a final volume of approximately 1 mL.

Recombinant interferon gamma in so-called single chain form (IFNγSC) described by [25 (link)] was cloned into pET-26b(+) vector (Novagen) using NdeI and XhoI restriction enzymes in frame with N-terminal start codon not to have no peptide leader nor tag.
The recombinant IFNγSC was expressed in E. coli BL21 (λDE3) in LB medium containing 60 μg/mL of kanamycin at 30°C for 4 hours after induction by 1 mM IPTG. Harvested cells by centrifugation (8,000 g, 10 min, 4°C) were disrupted by ultrasound in 20 mM Na-Phosphate buffer pH 7.3 and centrifuged at 40,000 g, 30 min, 4°C, and soluble fraction was further purified on SP Sepharose HP (GE Healthcare) using linear gradient of NaCl and further purified to homogeneity by gel filtration in same procedure as IFNγR1 receptor (see above).

For purification of Alp14-GFP, 10–20 ml of cleared lysate was loaded on to a 1.2 ml SP Sepharose HP (GE Healthcare) column equilibrated with low-salt cation exchange buffer. The resin was washed with 7.5 ml low-salt cation exchange buffer and 10 ml of 250 mM NaCl in cation exchange buffer. Proteins were eluted with 10 ml of 500 mM NaCl in cation exchange buffer. Fractions containing the target protein were pooled and loaded onto 500 μl of Ni-NTA Superflow resin (Qiagen) equilibrated with low-imidazole buffer. The resin was washed with 10 ml of low-imidazole buffer then imidazole buffer containing 30 and 60 mM imidazole buffer before elution with 500 mM imidazole buffer. Peak fractions containing Alp14 were pooled, aliquoted, quick frozen and stored in liquid nitrogen.