Thermo sorvall st16r centrifuge

The model solution, containing 20 mg mL⁻¹ L-theanine and 10 mg mL⁻¹ caffeine, was prepared and adjusted to different acidity (pH 1–9) through an appropriate addition of 1.0 M HCl or NaOH. An amount of model solution (50 mL) was mixed with pretreated cation exchange resins (2.500 g equivalent to dry weight) and shaken at 25 °C and 150 rpm for 12 h in an HZ-9201K constant temperature oscillator (Taicang, China). The supernatant was obtained after the mixture was centrifuged at 6000× g and 25 °C for 10 min in a Thermo Sorvall ST16R centrifuge (Thermo Scientific Co., Rockford, IL, USA). The concentrations of L-theanine and caffeine in the supernatant were analyzed by a Shimadzu LC-20A HPLC (Shimadzu Corp., Kyoto, Japan). Test was repeated three times. The adsorption capacity and selectivity were calculated according to Equations (1) and (2).

Gene APASM_5716, coding for the cell division protein FtsZ in A. pretiosum WXR-24, was amplified by PCR using primers 5716-28a-FP and 5716-28a-RP (Table S1), sequenced and inserted into the NdeI/EcoRI sites of the vector pET28a. The resulted plasmid was introduced into E. coli BL21(DE3) for protein expression. The resulting strain was cultivated at 37 °C to OD_600nm of 0.6–0.8, then induced with 0.1 mM IPTG and cultivated at 30 °C for another 12 h. Cells were then harvested by centrifugation and re-suspended in 10 mM PBS (pH 7.5). After sonication, cell debris were removed by centrifugation at 11,000 rpm for 30 min on a Thermo Sorvall ST 16R centrifuge (ThermoFisher, Waltham, MA, USA). FtsZ was purified by nickel-NTA affinity chromatography after elution with PBS containing 50 mM–250 mM imidazole. The 250 mM imidazole eluent was ultra-filtrated into a volume of 500 μL, and then 10 mL corresponding buffer for the following assays was added, followed by ultra-filtration again into 500 μL. For SPR molecule interaction determination, the eluent after nickel-NTA purification was ultra-filtrated and directly injected into fast protein liquid chromatography (FPLC) equipped with a Superdex 200 gel filtration column (GE Healthcare Life Sciences, Marlborough, MA, USA) for further purification [19 (link)]. The purified proteins were used for analysis or stored at −80 °C for future use.