Purification of recombinant VEGF
b produced in Pichia pastoris. HPLC elution profile showing the absorbance overtime, after production of VEGF121b (A). Two peaks are observed for both isoforms: Peak 1 corresponds to proteins in the P. pastoris culture supernatants unbound to the column, whereas peak 2 corresponds to the eluted VEGF121b protein. Fractions indicated in the graphs were electrophoresed and stained with Coomassie blue. Bands of the expected size were observed in the fractions corresponding to peak 2 (B). Culture supernatants subjected to electrophoresis for both VEGF165b and VEGF121b. Under non-reducing conditions the 3-band pattern (light arrows) corresponds to dimers (most likely glycosylated-glycosylated, glycosylated-non-glycosylated and non-glycosylated-non-glycosylated proteins, as previously described for the VEGF-A classic isoforms). Under reducing conditions, bands (2-band pattern, as described for VEGF-A under these conditions) correspond to monomers (C). VEGF121/165b proteins purified after production in the yeast P. pastoris are strongly immunoreactive with an antibody raised against VEGFxxxb. The lanes in the blots show bands of 3 clones with different amounts of secreted VEGF121b, or 2 clones in the case of VEGF165b (D).