Bio rs 24

Powdered samples of various G. biloba L. plant parts were accurately weighed (30 mg) and sonicated for 10 min at room temperature in the presence of extraction solvent (1 mL of 80% methanol), followed by a 45 min rotation in a mechanical shaker (Bio RS-24, Biosan, Latvia) and 5 min centrifugation (LMC-4200R, Biosan, Latvia) at 4000× g. Prior to the instrumental analysis, the obtained sample extracts were filtered through a 45 µm pore size polytetrafluoroethylene syringe filter. Each plant sample type was prepared in triplicates after which the average result value was expressed for each biflavonoid compound.

For Sup35NM purification, pET-20b-SUP35NM-His₆ (Allen et al., 2005 (link)) plasmid or its derivative for Sup35NM-M0 overproduction were used. For protein purification, E. coli strain BL21(DE3) was used (Studier and Moffattf, 1986 (link)). Overproduction of recombinant proteins was carried out in 2TYa media with 1 mM IPTG. Cultures were grown at 37°C for 6 h. Proteins were purified in denaturing conditions (in the presence of 8 M urea) according to previously published protocols (Glover et al., 1997 (link); Serio et al., 1999 (link)). The purification was performed with a two-step procedure with Ni-NTA agarose (Invitrogen) and Q-sepharose (GE Healthcare) columns. Proteins were concentrated with a centrifuge concentrator with molecular weight cutoff of 30 kDa (Millipore).
The obtained Sup35NM proteins were diluted at least 100-fold into fibril assembly buffer (5 mM potassium phosphate pH 7.5, 150 mM NaCl) to a final protein concentration of 0.5 mg/ml. In these conditions, Sup35NM spontaneously forms aggregates. Samples were incubated at 26°C with slow overhead rotation (rotator Bio RS-24, Biosan). To monitor amyloid fibril formation, aliquots were removed every 12 h up to 24 h of incubation. The rate of aggregated protein was estimated by SDS-PAGE with boiled and unboiled samples.

E. coli strain BL21(DE3) [27 (link)] transformed with pDEST527-NUP58 was used for the protein purification. Overproduction of the recombinant protein was carried out in LB media with 100 μg/mL ampicillin and 1 mM IPTG. Cultures were grown at 37 °C for 6 h. NUP58 was purified in denaturing conditions in the presence of 8 M urea according to the protocols proposed for Sup35NM [28 (link)]. The purification was performed using only Ni-NTA agarose (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, R901-15). The obtained NUP58 protein was diluted at least 100-fold into fibril assembly buffer (5 mM potassium phosphate pH 5.8, 150 mM NaCl) to a final protein concentration of 1 mg/mL. At these conditions, NUP58 spontaneously forms aggregates. Samples were incubated at 37 °C with slow overhead rotation (rotator Bio RS-24, Biosan, Riga, Latvia). To monitor amyloid fibril formation, aliquots were removed every 12 h up to 24 h of incubation. The rate of aggregated protein was estimated by SDS-PAGE with boiled and unboiled samples. Sup35NM aggregates were obtained as described previously [25 (link)].