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).