Fig. 2
Small-scale screening of p-blinatumomab-expressing Pichia pastoris clone. (a) P. pastoris GS115 and KM71 clones transformed with the p-blinatumomab recombinant linearized plasmid DNA vector were cultured in BMMY medium with MeOH for 96 h (final MeOH concentration: 0.5%, 24-h intervals), and equal amounts (10 µL) of the extracted eluted fractions were analyzed on a 12% SDS-PAGE gel. Lane M, pre-stained protein marker (Kanpro Research. KP-PR001); Lanes 1 and 2, not transformed GS115 and KM71 (negative controls); Lane 3, GS115-HSA secreting human serum albumin (HSA) to BMMY medium (positive control); Lanes 4–13, 5 clones of GS115/p-blinatumomab (Clone number;1, 2, 3, 105, 108 and 5 clones of KM71/p-blinatumomab (Clones 1, 4, 91, 104, 108) purified by Ni-NTA resin affinity chromatography and analyzed on a 12% SDS–PAGE gel. The GS115/p-blinatumomab #105 clone producing the highest amount of p-blinatumomab was selected for further studies. Lane 14, 100 ng of BLINCYTO. HSA protein molecular weight is 63 kDa. (b) The expression rate of p-blinatumomab, quantified by UV-Vis spectrophotometry at 280 nm was determined as the total recovered purified protein (μg) per liter of culture medium (μg/L). All values are expressed as the mean ± standard deviation (SD) based on three independent experiments (n = 3).
