Human Pregnancy Specific Beta-1-Glycoprotein 1 (PSG1) Has a Potential Role in Placental Vascular Morphogenesis

Ha, Cam T.; Wu, Julie A.; Irmak, Ster; Lisboa, Felipe A.; Dizon, Anne M.; Warren, James W.; Ergun, Suleyman; Dveksler, Gabriela S.

Recombinant proteins were purified by affinity chromatography using the Ä KTAprime Plus system (GE Healthcare). PSG1-FLAG was dialyzed into 20 mM sodium phosphate buffer (pH 7.4) containing 20 mM imidazole (EMD Chemicals, Inc.) and purified using a HisTrap column (GE Healthcare). The obtained fractions were pooled, buffer-exchanged into PBS, applied to a column packed with anti-FLAG M2 agarose (Sigma), and eluted with 33 FLAG peptide (Sigma). PSG1-Fc and the FLAG-Fc were dialyzed into 20 mM sodium phosphate buffer (pH 7.4) and purified using a HiTrap protein A column (GE Healthcare). The proteins were eluted with 0.1 M glycine (pH 2.7) and collected into tubes containing 100 ll of 1 M Tris-HCl (pH 8.0). Fractions containing the purified PSG from the anti-FLAG agarose or protein A columns were identified by Western blot analysis with the anti-PSG1 MAb BAP1 and were pooled. The pooled fractions were concentrated and buffer-exchanged with PBS using Amicon Ultra-15 10-kDa MWCO centrifugal filter units (Millipore Corp.). The purified proteins were run on a SDS-PAGE gel, stained with GelCode Blue Stain Reagent (Pierce), and quantitated against bovine serum albumin standards. In addition, the recombinant PSG1 proteins were immunoblotted with anti-FLAG (for PSG1-FLAG and FLAG-Fc) and antihuman Fc (for PSG-1Fc, the PSG1 mutants, and FLAG-Fc) after separation using SDS-PAGE. In all cases, 500 ng of protein were loaded per lane, and the antibodies were used at a concentration of 1 lg/ml overnight in 5% milk in Tris-buffered saline Tween-20 and were followed by a 1:10 000 dilution of the horseradish peroxidase-labeled secondary antibody (Bio-Rad).