Bradford protein assay reagent

E. coli BL21(DE3) harboring pETDuet-Bga1903+ was grown aerobically in LB, supplemented with 100 μg/mL ampicillin, at 37 °C to an OD₆₀₀ ≈ 0.8. At the time, IPTG was added into the culture to a final concentration of 1 mM and the cultivation was continued at 28 °C for 18 h. The culture was centrifuged at 10,000× g for 15 min at 4 °C, and the cell-free supernatant was harvested. This protein solution was 100-fold concentrated by using the Millipore Labscale TFF system, equipped with Pellicon XL cassette (Biomax 5 kDa). The concentrated solution was loaded into a HisTrap excel 5 mL column (GE Healthcare Life Science, Marlborough, MA, USA), followed by an intensive wash with PBS buffer (20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4). Finally, the protein bound on the resin was eluted with 100 mM imidazole-containing PBS buffer. The concentration of the purified protein was measured using the Bradford protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA) with BSA as the standard.

Recombinant Bga1903 was produced and purified as described previously [21 (link)]. In brief, E. coli BL21(DE3) pLysS was cultured overnight with Lysogeny broth (LB, containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl). Fresh LB medium containing 100 μg/mL ampicillin inside a shake flask was inoculated with overnight bacterial culture, and placed inside a shaking incubator at 37 °C at 200 rpm until the OD₆₀₀ reached approximately 1.0. Administrated with 1 mM final concentration of isopropyl β-D-1-thiogalactopyranoside, the cell culture was incubated for 18 h at 28 °C at 200 rpm. To harvest secreted Bga1903 within the medium, the bacterial broth was centrifuged at 10,000× g for 15 min at 4 °C, and the cell pellet was discarded afterward. Bga1903 inside the culture medium was harvested and subsequently purified by using a column packed with Qiagen Ni-NTA agarose resin (Venlo, The Netherlands). The purified protein was obtained by concentrating elution fractions using a 10 kDa Amicon ultra-4 centrifugal filter unit (Merck, Darmstadt, Germany), and preserved in the buffer containing 20 mM Tris [pH 8.0] and 500 mM NaCl. The concentration of the purified protein was measured using Bradford protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as the standard.