Figure 5

SDS-PAGE analysis of secretory expression of NFG1CG4-hFasLECD. a) Comparison of the expression level using glass baffled-flask with that using plastic culture-bag. Lanes: M, molecular-weight size-markers; a and b, at 0 h; c and d, at 24 h; e and f, at 48 h; g and h, at 72 h; i and j, at 96 h. Five μl each samples of culture supernatants either from 500 ml scale culture in 3000 ml baffled flask (lanes a, c, e, g and i) or 2500 ml scale culture in 5000 ml disposable plastic bag (lanes b, d, f, h and j) were applied to each lane. Upper and lower arrows indicate the migration positions of dimeric and monomeric subunits of NFG1CG4-hFasLECD, respectively. b) SDS-PAGE analysis of disulfide bond reduction in partially purified NFG1CG4-hFasLECD. Main peak fraction of the P. pastoris culture supernatant (29.5°C, 96 h) in 1st cation-exchange column chromatography was concentrated and treated with SDS-PAGE sample buffers with / without 2-mercaptoethanol. Lanes: M, molecular-weight size markers; a, with 2-mercaptoethanol; b, without 2-mercaptoethanol. Upper and lower arrows indicate the same as in a). c) Co-immunoprecipitation of the secreted product with wild-type hFasRECD-T-Fc. Lanes: M, molecular-weight size-markers; a, 5 μl culture supernatant of 40-fold concentrated NFG1CG4-hFasLECD from 500 ml scale culture in 3000 ml baffled flask at 96 h; b, purified NFG5-hFasLECD [10] (5 μg); c, buffer alone. Upper and lower arrows indicate the same as in a). The bands in “FasR-Fc” labeled region were derived from wild-type hFasRECD-T-Fc sample.