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. Author manuscript; available in PMC: 2008 Dec 4.
Published in final edited form as: Biotechnol Prog. 2007 Feb 22;23(2):364–369. doi: 10.1021/bp060287p

Figure 4.

Figure 4

Overexpression of PDI does not increase the secretion of either wild-type or C75S β-glucosidase at 37°C where the ER secretion bottleneck is removed as detected by activity assay. Single colonies of BJ wt, hPDI wt, BJ C75S, and hPDI C75S were grown for 48 h in 5 mL SD-2xSCAA dextrose liquid growth media minus tryptophan supplements at 30°C. SD media was removed by centrifugation, and the cell pellet was resuspended to 1 OD-mL in 5 mL SG-2xSCAA galactose liquid expression media minus tryptophan supplements. After expression for 48 h at 37°C, extracellular β-glucosidase levels were detected in a constant volume of supernatant separated from cells by centrifugation by Western blot analysis (filled bars) using an anti-c-myc polyclonal antibody for each expression and normalized per cell OD. 20 μL of the supernatant was separated by centrifugation and assayed for active extracellular β-glucosidase (open bars), normalized per cell OD. The average of six independent expressions is plotted, and the error bar represents the standard error.