Benzamidine sepharose 4 fast flow resin

Benzamidine sepharose 4 fast flow resin (GE healthcare # 17-5123-10) was used to purify serine protease enzymes from the M44 culture supernatant sampled after 9 days. 15 ml of the fermentation culture supernatant was batch bound to the resin in 35 ml of binding buffer (0.05 M Tris-HCL, 0.5 M NaCl, pH 7.4). After packing and washing the column with the same binding buffer, the column was eluted with 0.05 M glycine, pH 3.0. The fractions were then neutralized with 1M Tris HCL, pH 8.8. The peak fraction protein mixture (F4) and IgG were incubated together with and without PMSF in sodium citrate buffer (50 mM, pH 5.5) at 37°C for 20 hours. The nonreduced samples were loaded into a 12% SDS PAGE gel and an immunoblot with heavy and light chain AP conjugated antibodies was done to analyze the reaction samples.

The cloned mMT-I/pGEX-4T-1 plasmid and mouse β₂-MG/pGEX-4T-1 plasmid were transformed into BL21(DE3)pLysS (Promega, Madison, WI, USA). The selected transformed cells were grown in 10 mL SOB medium containing 50 μg/mL ampicillin for 16 h at 37 °C until the optical density at 600 nm reached 0.3–0.4. The expression of MT and β₂-MG proteins was induced by incubation with 1 mM IPTG for 6 h at 37 °C. The cultured cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C, and the GST-fusion proteins were purified by using MagneGSTTM Protein Purification (Promega). After a dialysis against PBS, the GST-fusion MT and GST-fusion β₂-MG proteins were digested with thrombin (GE Healthcare, Buckinghamshire, UK). The lysates were loaded onto GST GraviTrapTM gravity-flow columns (GE Healthcare) to remove GST proteins, and the lysates were loaded onto a Benzamidine Sepharose 4 Fast Flow resin (GE Healthcare) to remove thrombin.
The purified recombinant MT and β₂-MG proteins were conjugated with fluorescein isothiocyanate (FITC) by Fluorescein Labeling Kit-NH2 (Dojindo, Kumamoto, Japan). For FITC-labeled albumin and Alexa-labeled transferrin, commercially available albumin-fluorescein isothiocyanate conjugate and Alexa Fluor^® 488-conjugated ChromPure Mouse Transferrin, respectively, were used.