Expression and purification of recombinant GFAPs

For bacteria expression of GFAP, pET23b vector containing either WT or mutant GFAPs was transformed into the Escherichia coli BL21 pLysS strain (Agilent Technologies, Santa Clara, CA). Induction of recombinant protein overexpression was achieved by the addition of 0.5 mM Isopropyl 1-thio-β-d-galactopyranoside (IPTG) when the optical density (OD₅₉₅) reached 0.2. After incubation for an additional 4 h, bacteria were harvested by centrifugation at 6000 × g for 30 min at 4°C. Overexpressed GFAP formed inclusion bodies, which were prepared as described previously (Perng et al., 2006). The final pellets, consisting predominantly of GFAP, were extracted in urea buffer (6 M urea, 20 mM Tris-HCl [pH 8], 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) at 4°C overnight. After centrifugation at 80,000 × g for 20 min at 4°C, the urea-soluble fractions were treated with 0.05% (vol/vol) polyethyleneimine (PEI; Sigma-Aldrich, St. Louis, MO) to precipitate contaminating bacterial DNA. After centrifugation under the same conditions, GFAP in the supernatant was further purified by anion-exchange chromatography using an AKTAprime plus system (GE Healthcare, Chicago, IL). GFAP was eluted from a DEAE Sepharose (GE Healthcare) column with a linear gradient of 0–1 M NaCl in the urea buffer over 1 h at a flow rate of 1 ml/min. Column fractions were analyzed by SDS–PAGE followed by Coomassie brilliant blue staining, and those containing purified GFAP were pooled and stored at −80°C. The protein concentration was determined by bicinchoninic acid assay (BCA) assay (Thermo Fisher Scientific, Waltham, MA) using bovine serum albumin (BSA) as a standard.