Protein solubility and isolation
Seed storage proteins : Aqueous meal extracts were prepared at pH values between 2 and 12 according to Perera et al. (2016). The soluble protein content of extracts was determined from nitrogen content as above. The polypeptide profile of the soluble protein at each pH was evaluated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below. Cruciferin and napin were extracted from mucilage-reduced, oil-free meal and purified following the processes described by Perera et al. (2016) for B. napus . Purified cruciferin and napin fractions were lyophilized, analyzed for total protein content and stored at -20°C.
Oil body proteins (OBPs) : Oil bodies (OBs) were separated from mucilage-free seeds according to Maurer et al. (2013) with modifications. Seed was soaked overnight in Milli Q water (1g seed:4 mL water) at 4°C, homogenized at 10,200 rpm for 1 min using a Polytron PT3100 homogenizer with PTDA 3020/2T generator (Kinematica Inc.) and filtered through three layers of cheesecloth to obtain a filtrate with OBs. The seed residue was re-extracted with deionized water (1:4, w:v) two more times, the filtrates combined, and sucrose added to achieve 25% (w:v) final concentration. The top cream layer containing OBs was recovered by centrifugation at 5,000 × g for 30 min, mixed with 20% (w:v) sucrose solution at pH 11 and then centrifuged again. The OB layer was recovered and dispersed in 100 mL of deionized water and dialyzed (2 kDa MW cut-off) against deionized water for 24 h at 4°C. The dialysate was centrifuged and the supernatant mixed with 1% SDS (10:1, v:v, dialysate-to-SDS solution). The aqueous medium containing OBPs was recovered by centrifugation (7500 × g for 30 min) and then dialyzed (2 kDa MW cut-off) for 24 h against deionized water at room temperature. Lyophilized OBPs were reconstituted in deionized water and separated on a HiPrep 26/10 desalting column with deionized water as the mobile phase with detection at 280 nm (ÄKTA Explorer system, GE Healthcare Life Sciences).