Figure - PMC

Skip to main content

View full-text article in PMC

. 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

Time related overexpression of PDI at 30°C shows increased secretion of both wild-type and C75S β-glucosidase after 24 h until the final time measured at 96 h. Strains grown for 48 h in 25 mL SD-2xSCAA dextrose liquid growth media minus tryptophan supplements with 1 mg/mL BSA were separated from SD media by centrifugation and diluted to 1 OD-mL in 25 mL SG-2xSCAA galactose liquid expression media minus tryptophan supplements. At each time point, a supernatant fraction was separated by centrifugation and a constant volume was assayed for secreted β-glucosidase by activity. Additionally, 1 OD-mL equivalent was collected and intracellular β-glucosidase and intracellular PDI were detected by Western blotting with anti-c-myc polyclonal or anti-PDI monoclonal antibodies. a) Secreted wild-type and C75S β-glucosidase measured by activity assay of supernatant fractions, with filled circles representing BJ wt, open circles hPDI wt, filled square BJ C75S, and open squares hPDI C75S, normalized per cell OD. Data represents average of two independently selected transformants. b) Western blot detection of PDI and β-glucosidase in the intracellular fraction over 96 h time course. Lanes marked 1-4 correspond to a single transformant each of BJ wt, hPDI wt, BJ C75S, hPDI C75S, respectively at 24 h, 48 h, 72 h, and 96 h, loaded in equal OD equivalent volumes.

Time related overexpression of PDI at 30°C shows increased secretion of both wild-type and C75S β-glucosidase after 24 h until the final time measured at 96 h. Strains grown for 48 h in 25 mL SD-2xSCAA dextrose liquid growth media minus tryptophan supplements with 1 mg/mL BSA were separated from SD media by centrifugation and diluted to 1 OD-mL in 25 mL SG-2xSCAA galactose liquid expression media minus tryptophan supplements. At each time point, a supernatant fraction was separated by centrifugation and a constant volume was assayed for secreted β-glucosidase by activity. Additionally, 1 OD-mL equivalent was collected and intracellular β-glucosidase and intracellular PDI were detected by Western blotting with anti-c-myc polyclonal or anti-PDI monoclonal antibodies. a) Secreted wild-type and C75S β-glucosidase measured by activity assay of supernatant fractions, with filled circles representing BJ wt, open circles hPDI wt, filled square BJ C75S, and open squares hPDI C75S, normalized per cell OD. Data represents average of two independently selected transformants. b) Western blot detection of PDI and β-glucosidase in the intracellular fraction over 96 h time course. Lanes marked 1-4 correspond to a single transformant each of BJ wt, hPDI wt, BJ C75S, hPDI C75S, respectively at 24 h, 48 h, 72 h, and 96 h, loaded in equal OD equivalent volumes.