2S albumin family, PFAM PF00234; Tryp_alpha_amyl

http://us.expasy.org/cgi-bin/niceprot.pl?P19594

12 kDa (Gu et al. 2000 [654])
14 kDa (Ogawa et al. 1991 [441]; Herian et al. 1990 [1078])

8975.14 and 5138.30 (Lin et al. 2004 [1077])

(Lin et al. 2004 [1077]) reported the purification of both native soybean 2S albumin from soybean and recombinant soybean albumin, rAL, from Pichia pastoris.

Native soybean 2S albumin:
Soybean seeds were ground into a fine powder, defatted by extraction with hexane (1:4 w/v) and dried in vacuum. Soluble proteins were extracted from 100 g of defatted soybean powder with 400 ml albumin extraction buffer (10% v/v DMSO, 0.5% v/v butan-1-ol, 100 mM KCl, 83 mM sodium acetate, pH 5.2). Following centrifugation at 6000×g for 15 min, the supernatant was dialyzed against 0.5% v/v butan-1-ol, 100 mM KCl, 83 mM sodium acetate buffer, pH 5.2, lyophilised, and dissolved in distilled water (250 mg/ml). Insoluble proteins were removed by centrifugation at 6000×g for 15 min and the supernatant was loaded onto a Sephacryl S-100 column (2 cm×55 cm, Pharmacia) run (0.5 ml/min) with the albumin extraction buffer. Fractions containing the 2S albumins (~14 kDa) were dialyzed against distilled water, lyophilised, dissolved in 20 mM Tris/HCl, pH 8.5 (20 mg/ml) and further fractionated by ion-exchange chromatography using the BioCAD perfusion chromatography system equipped with a POROS 20 HQ (4.6 mm×10 cm) column, eluted with a gradient of NaCl (0750 mM) in 20 mM Tris/HCl, pH 8.5 at a flow rate of 5 ml/min. The peak, which was eluted at 360 mM NaCl and contained the 2S albumin, was then further purified by reversed-phase HPLC on a C₁₈ column (25 cm×10 mm, Phenomenex), in 0.07% (v/v) trifluoroacetic acid (TFA) in water and eluted with a linear gradient of 050%, 100% acetonitrile in 0.05% (v/v) TFA at a flow rate of 2.5 ml/min for 40 min and the purified 2S albumin eluted after 25 min. The eluted peak was lyophilised and stored at −20 °C.

Recombinant soybean albumin:
The supernatant secreted by P. pastoris was filtered through a 0.45µm filter, then concentrated and salt eliminated by buffer exchange with 10 mM citric acid buffer (pH 4.3) using a Viva Flow 200 (5000 MWCO) (Vivascience, Lincoln, UK). The concentrate was loaded onto three in-series 5 ml Hi-Trap heparin columns (Amersham-Pharmacia, Uppsala, Sweden) previously equilibrated with 10 mM citric acid buffer (pH 4.3) and eluted using a step gradient of NaCl (0.21.0 M) at a flow rate of 3 ml/min. The column effluent was monitored at 280 nm. The collected fractions containing rAL were dialyzed against distilled water for 24 h at 4 °C. The rAL was then further purified by reversed-phase HPLC on a C₈ column (10 cm×10 mm, Varian, Walnut Creek, CA, USA), in 0.1% (v/v) formic acid in water and eluted with a linear gradient of 0100% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 2.5 ml/min for 25 min. The eluted peak was lyophilised and stored at −20 °C.

The sequences P19594 and AAD00178.1 are identical. A further sequence AAD09630 is related but distinct. Both albumins were purified by Lin et al. (2004) [1077].

Gu et al. (2000) [654]) reported that the 2S albumin was strongly bound at 12 kDa by IgE binding from a single sera and weakly by IgE from another sera. The 14 kDa minor allergen reported by Ogawa et al. (1991) [441] and Herian et al. (1990) [1078] may also be the 2S albumin. The reported masses are high as the proteins were reduced before electrophoresis.