Sephacryl s 100 column

To separate and fractionate the components of the crude bee venom, a gel filtration chromatography technique was used. The crude sample (150 mg) was dissolved in 3 ml of 50 mm phosphate buffer pH 7.2, which was then centrifuged at 10,000 rpm for 20 min to remove the impurities. The filtered sample of bee venom was applied to a Sephacryl S100 column (16/60, 120 ml, GE Health care, Sweden) previously equilibrated with the same buffer. Fractions were eluted with 50 mm phosphate buffer pH 7.2 containing 0.15 M NaCl using FPLC (AKTA Prime plus, GE Healthcare, Sweden) at a flow rate of 0.5 ml/min and fraction size of 4 ml/fraction. The optical density of eluted fractions was measured at 280 nm and the molecular weight of the crude sample and eluted fractions were estimated by SDS-PAGE.

The ETR1-RD and its mutant cDNA fragments cloned in pET6H were transformed into E. coli strain BL21 (DE3) and grown in LB medium with 0.1 mg mL^-1 ampicillin. The cells were cultured at 37°C until the OD₆₀₀ reached 0.6 and were subsequently induced by adding isopropyl-1-thio-β, D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM for 20 hours at 16°C. The cells were harvested by centrifugation, resuspended in lysis buffer (40 mM Tris-HCl, pH 8.0 and 150 mM NaCl), and lysed using a high-pressure homogenizer (GW technologies, Taiwan). The 6×His-tagged ETR1-RD was purified by affinity chromatography using Ni⁺-NTA agarose (GE Healthcare). The proteins were further purified by size-exclusion chromatography using a Sephacryl S-100 column (GE Healthcare) in FPLC buffer (40 mM Tris-HCl, pH 7.0, 150 mM NaCl and 1 mM EDTA). The purified proteins were verified by mass spectrometry. The protein concentration was estimated based on the extinction coefficient and absorption at 280 nm via Nanophotometer (IMPLEN). To prepare the ¹⁵N-labeled or ¹⁵N-, ¹³C-labeled samples for NMR, the cells were grown in M9 minimal medium supplemented with ¹⁵NH₄Cl (1 g L^-1) and ¹³C _D-glucose (2 g L^-1) as the sole nitrogen and carbon source.

E. coli BL21star(DE3)pLysS cells transformed with the Pro21717-PCD construct were grown at 37°C in 1 L of LB medium supplemented with ampicillin (100 μg/mL). After growing to an OD₆₀₀ of 1.0, expression was induced with 1.0 mM IPTG at 37°C for 24 h. The cell pellet was obtained by centrifugation (10,000 × g, 30 min, 4°C), resuspended in 4 mL standard buffer (50 mM sodium phosphate, pH 7.6), and sonicated. After centrifugation (10,000 × g, 30 min, 4°C), the resulting insoluble fraction was washed twice (10,000 × g, 10 min, 4°C) with 20 mL of 0.5% Triton X-100. The washed inclusion body (1 g) was unfolded in 20 mL unfolding buffer (pH 8.5; 8 M urea, 50 mM mercaptoethanol, and 20 mM Tris-HCl) at 37°C for 1 h. The unfolded protein solution was diluted in 60 mL refolding buffer (20 mM Tris-HCl, 100 mM NaCl, 20 mM CaCl₂, and 0.05% Tween 20) and subsequently dialyzed in 1.6 L refolding buffer at 4°C for 2–3 days. The refolded protein solution was loaded onto a Sephacryl S-100 column (1.6 mm × 70 cm, GE Healthcare) in a Dual Flow FPLC instrument (Bio-Rad). The proteins were separated according to size in the mobile phase (standard buffer) at a flow rate of 0.7 mL/min. A fraction with significant proteolytic activity was concentrated and used as a homogenous enzyme solution (designated as Pro21717-CD) in subsequent experiments for biochemical characterization and crystallization.