Figure 3

Secretion of recombinant Bm86, Bd86 and Ba86 by P. pastoris. Silver stained SDS-PAGE (lanes 1–5) and Western blot analysis (lanes 6–10) of the fermentation culture supernatants after 72 hrs growing in methanol. Samples of 15 μL were loaded in each well. Membranes for Western blot were probed with serum from rabbits immunized with control Bm86 (Gavac; Revetmex) diluted 1:1000. Membranes were then washed three times with TBS and incubated with an anti-rabbit IgG HRP conjugate (Sigma-Aldrich) diluted 1:1000 in TBS. After washing the membranes again, color was developed using TMB stabilized substrate for HRP (Promega). Lanes 1 and 6: molecular weight markers (MW; ColorBurst, Sigma-Aldrich). Lanes 2 and 7: culture supernatants of the P. pastoris GS115/Albumin negative control strain. Lanes 3 and 8, 4 and 9, and 5 and 10: culture supernatants of X33pBaI-3 (Ba86), GS115Moz9-2 (Bm86) and KM71HDec8-1 (Bd86) strains, respectively. The position of recombinant proteins is indicated with arrows.