Gstrap 4b

pGEX-4T-3-LRB7 plasmid was built by combining LRB7 coding regions and PGEX-4T-3 plasmid and introduced into E. coli BL21 (DE3) pLysS. The transformed E. coli BL21 (DE3) pLysS was cultured in lysogeny broth (LB) medium for overnight. Then, 0.4 mL of liquid bacteria was cultured in 20 mL LB medium until the optical density (OD) reached 0.6 at 600 nm. By adding 24 µL of 20% isopropyl-β-D-thiogalactoside (20% IPTG), fusion proteins (GST-LRB7) were induced for 6 hr from 1 liter of liquid transformed bacteria, obtained by boiling for 12 min, separated by 12% SDS-PAGE gels, and purified by using GSTrap 4B (GE Healthcare Life Sciences) following the protocols. Next, purified GST-LRB7 proteins (100 µg) and Complete Freund's Adjuvant (equal volume to GST-LRB7 protein medium, Sigma-Aldrich) were used to simultaneously immunize two Japanese adult male rabbits every two weeks. Blood serum of the rabbits was harvested and polyclonal antibody was purified by Protein A Sepharose (CL-4B) following standard protocol (Invitrogen) and purified by using affiliation column with bound GST to remove the anti-GST antibody. The purified anti-LRB7 was stored at −80°C in a buffer containing 1% BSA, 50% glycerol, and 0.02% sodium azide for further use.

Expression of recombinant full-length human PAH construction (PAHt) and double truncated form (PAHd) and their purification were performed as described in [38 (link)]. PAHt and PAHd were expressed as maltose-binding fusion proteins in E. coli BL21 (DE3) cells grown in Super Broth medium with 100 µg/mL of ampicillin at 37 °C. Expression was induced by 1 mM IPTG, and the culture was prolonged at 20 °C for two days. Recombinant MBP-PAH proteins were purified using amylose affinity chromatography followed by gel filtration on Superdex 75 XK26/60. Collected fractions were cleaved overnight with PreScission Protease at 4 °C and the untagged proteins were further purified using two affinity columns, MBPTrap HP and GSTrap 4B (GE Healthcare). A second size exclusion chromatography step was then used to isolate the tetrameric form of PAHt and the dimeric form of PAHd. The proteins were concentrated and their final concentrations were determined spectrophotometrically using theoretical extinction coefficients at 280 nm [39 ].