Fig 1.
Time-course profiles of rDM64 secretion in Pichia pastoris culture during fermentation.
P. pastoris was grown at 30°C and 250 rpm for 288 hours. 0–24 hours: glycerol batch phase. 24–288 hours: methanol induction (fed-batch phase); 1% methanol was added every 24 hours, for 264 hours. Dry cell weight/biomass (circle) and rDM64 concentration (square) were determined in the absence (A) or presence (B) of 0.2 mM Pefabloc. The concentration of rDM64 was measured by immunoassay in the culture medium and was performed in triplicate. Error bars represent the standard deviation of the mean.
Fig 2.
SDS‑PAGE (12%) analysis of culture supernatant from P. pastoris fermentation during the growth time in the absence (A) or presence (B) of 0.2 mM Pefabloc. Aliquots of P. pastoris culture media (~ 14 μL) that were taken at different time points were loaded into each lane under reducing conditions. The gels were silver stained, and the proteins in the gel bands that were indicated by red rectangles were identified by MS/MS (S1 Table). (C) Identification of rDM64 in yeast culture medium by immunoblotting using polyclonal antibodies raised against native DM64. Different induction times were assayed in the absence of Pefabloc. The asterisk indicates native DM64 purified from opossum serum, which was used as the positive control. MM represents prestained molecular mass markers.
Fig 3.
Characterization of rDM64 protein purified from yeast medium.
(A) Purification of rDM64 from the culture medium supernatant by affinity chromatography with immobilized myotoxin II. (B) After elution, the bound fraction (indicated by an asterisk) was further purified by anion-exchange chromatography using a Mono Q column. The chromatographic fractions were analyzed by 12% SDS-PAGE under reducing conditions and were stained with Coomassie blue (C) and periodic acid-Schiff reagent (D) or by immunoblotting with polyclonal antibodies raised against DM64 (E). Lane 1, native DM64 (5 μg). A typical degradation product ~ 45 kDa in size, generated by sample manipulation, can also be observed; lane 2, crude culture medium. Asterisks indicate the chromatographic fractions and are shown in panels A and B (5 μg/lane). NG, non-glycosylated protein (soybean trypsin inhibitor) used as negative control for the periodic acid-Schiff staining. MM, molecular mass markers.
Fig 4.
Enzymatic deglycosylation of DM64 by PNGase F analyzed by SDS-PAGE.
Native DM64 and affinity-purified rDM64 proteins were denatured before the removal of the carbohydrate residues. The glycoproteins were incubated with 1,000 U of recombinant PNGase F at 37°C for 1 hour. Lane 1, native DM64 before deglycosylation; lane 2, native DM64 after deglycosylation; lane 3, rDM64, purified by affinity column, before deglycosylation; lane 4, rDM64, purified by affinity column, after deglycosylation. The asterisks indicate protein bands corresponding to the PNGase (36 kDa). Samples were analyzed under reducing conditions on 12% gels that were stained with silver nitrate.
Fig 5.
Complex formation between myotoxin II (Lys49-PLA2) from B. asper venom and anion-exchange purified rDM64.
Myotoxin II (mt II) and rDM64 were mixed at a 2:1 (mol:mol) ratio and incubated for 30 minutes at 37°C; this sample was then analyzed using 12% native PAGE stained with silver nitrate. Lane 1, native DM64 (2.3 μg); lane 2, mt II-native DM64 complex (2:1, mol:mol); lane 3, mt II (1 μg); lane 4, rDM64 (2.3 μg); and lane 5, mt II-rDM64 (2:1, mol:mol).
Fig 6.
Inhibitory activity of rDM64 purified by affinity chromatography on the cytotoxic activity of B. asper myotoxin II upon C2C12 myotubes.
Myotoxin II (mt II) was incubated with native DM64 or rDM64 for 30 minutes at 37°C. The mixture was then added to a C2C12 myotube culture. Cytolysis was monitored based on the amount of lactate dehydrogenase that was released into the medium 3 hours after the addition of myotoxin alone (black) or myotoxin in complex with native DM64 (light grey) or rDM64 (dark grey). The bars represent the mean ± SD of triplicate cell cultures. * 0.01 < P < 0.05 and *** P < 0.001 when compared to the myotoxin control. The inhibition effects of equivalent doses of native and recombinant DM64 were also compared (# 0.01 < P < 0.05). Statistical analyses were by one-way ANOVA followed by Student–Newman–Keuls post hoc test.