Fast protein liquid chromatography system

The crude extract was prepared according to previous methods [30 (link)]. An algal sample (30 g) was immersed in liquid nitrogen and ground to a fine powder with a mortar and pestle. Five volumes of extraction buffer (Tris-buffered saline (TBS): 20 mM Tris-Cl, 150 mM NaCl, pH 7.5) were added to the sample to prepare the crude extract. The sample was incubated for 2 h at 4 °C, centrifuged at 20,000× g for 20 min at 4 °C and then the supernatant was collected as the crude extract. Then, D-mannose (Man) chromatography was immediately performed on the crude extract using a Bio-rad fast protein liquid chromatography system (Bio-rad, Berkeley, CA, USA). The column was washed with 10 volumes of TBS. Mannose-binding proteins were eluted with 0.5 M D-mannose with an extraction buffer by monitoring the absorbance at 280 nm. The fractions showing single bands following sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were fooled. The purified protein was dialyzed in TBS buffer overnight with buffer changes every 4 h. The total protein and purified protein concentrations were measured by a Bradford micro-assay [31 ] using an enzyme-linked immunosorbent assay (ELISA) reader (Epoch microplate spectrophotometer, BioTek, Winooski, VT, USA).

The harvested E. coli cells were resuspended in 50 mM citrate/phosphate buffer at pH 5.5 and pH 7.0 containing 300 mM NaCl. The resuspended cells were disrupted by sonication using a Sonic Dismembrator (Fisher Scientific Model 100, Pittsburgh, PA, USA) on ice for 10 min. The unbroken cells and cell debris were removed by centrifugation at 13,000×g for 20 min at 4 °C, and the supernatant was used as a crude extract. The crude extract in 50 mM citrate/phosphate buffer (pH 5.5) was subsequently heated at 75 °C for 10 min. After heat treatment, the suspension was centrifuged at 13,000×g for 20 min to remove insoluble denatured proteins. The supernatant obtained was used as the partially purified enzyme. The crude extract in 50 mM phosphate buffer (pH 7.0) was applied to a His-trap HP affinity chromatography column (GE Healthcare, Piscataway, NJ, USA). The bound protein was eluted with a linear gradient of 10–250 mM imidazole with 50 mM phosphate buffer (pH 7.0) at a flow rate of 1 ml min⁻¹. The active fractions were collected and dialyzed against 50 mM citrate/phosphate buffer (pH 5.5) at 4 °C for 16 h. The resulting solution was used as the purified enzyme. The purification step with the column was carried out in a cold room at 4 °C with a fast protein liquid chromatography system (Bio-Rad, Hercules, CA, USA).

The cultured cells were harvested by centrifugation at 6000×g for 20 min at 4°C and washed twice with 0.85% (w/v) NaCl. The cells were disrupted by sonication on ice for 2 min in 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl and 1 mg/ml lysozyme. The unbroken cells and debris were removed by centrifugation at 13000×g for 20 min at 4°C, and the obtained supernatant was used as a crude extract. Purification was carried out using a fast protein liquid chromatography system (Bio-Rad) in a cold room at 4°C. The crude extract was applied to a His-Trap affinity chromatography column (GE Healthcare, Piscataway, NJ, USA) equilibrated with 50 mM Tris-HCl buffer (pH 8.0). The bound enzyme was then eluted at 4°C with the same buffer containing 250 mM imidazole at a flow rate of 1 ml/min. The active fractions were collected and dialyzed at 4°C for 16 h against 50 mM Tris-HCl buffer (pH 8.5). The resulting solution was used as the purified enzyme.