Purification of egg white heparin-binding proteins

Egg whites were collected from freshly laid eggs (Isa-Hendrix, St Brieuc, France) and were homogenized using an Ultra-Turrax homogenizer (T18 basic ULTRA-TURRAX, IKA-Werke, Staufen, Germany), sampled and kept frozen until further use.

Heparin-Sepharose chromatography using the batch method was performed according to manufacturer’s instructions. Briefly, egg white (see above) was diluted 1:1 in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 and incubated with heparin-Sepharose beads (10:1, v/v) overnight at 4 °C under constant but slow agitation. The next day, the beads were washed extensively with 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 until the absorbance at 220 nm reached zero, and were loaded onto a polypropylene column (QIAGEN, Courtaboeuf, France). Elution of bound proteins was achieved with 50 mM Tris-HCl, 1 M NaCl, pH 7.4. Eluted fractions were concentrated (Ultracel-3K, Merck Millipore, Molsheim, France) and injected on a gel filtration column (Hiprep 16/60 Sephacryl S-100 High Resolution, GE Healthcare Life Sciences, Velizy-Villacoublay, France) using 50 mM Tris-HCl, 300 mM NaCl, pH 7.4, as the mobile phase. Proteins composing each fraction and purified proteins were analyzed by 12.5% SDS-PAGE under non-reducing conditions followed by Coomassie Blue staining. Major peaks were collected and concentrated by ultracentrifugation, as described above. Beta-microseminoprotein-like (gi|513191195) was further purified by gel filtration. Vitelline membrane outer layer protein 1 (gi|268370086) and pleiotrophin (gi|444741724) were purified from the salt-soluble part of the vitelline membrane, successively by heparin-affinity chromatography, gel filtration and reverse-phase chromatography as described previously for AvBD11¹⁶. Purified AvBD11 and OVAX were obtained as previously described^16,17. The protein concentrations of OVAX (Ext. coefficient E1% = 9.11), AvBD11 (Ext. coefficient E1% = 15.86), VMO-1 (Ext. coefficient E1% = 21.13), pleiotrophin (Ext. coefficient E1% = 15.87) and beta-microseminoprotein-like (Ext. coefficient E1% = 15.18) were measured using absorbance at 280 nm and their respective E1% values (Nanodrop, ND-1000 Spectrophotometer, Wilmington, USA). The purity of purified proteins was systematically verified by SDS-PAGE and mass spectrometry.