FIG. 4.

scFv expression in a high-density Q-cell culture. (A) W3110 hns-205/pCMT2b-scFv/pRcd1 (Q-cell culture) was grown initially at 30°C, and when the culture reached an OD₆₀₀ of 25, a feed of yeast extract (10%) and either glucose (50%; diamonds) or glycerol (50%; squares) was initiated. A phased temperature shift (see text) was started at t = 0 (OD₆₀₀ of 35), and culture density was monitored over the next 24 h. Culture samples were taken at intervals and separated into supernatant and whole-cell fractions for further analysis. (B) scFv activity of supernatant (diamonds) and whole-cell (squares) fractions of the glucose-fed Q-cell culture. (C) scFv activity of supernatant (diamonds) and whole-cell (squares) fractions of the glycerol-fed Q-cell culture. (D) Whole-cell and supernatant fractions from the glycerol-fed culture were separated by SDS-PAGE, stained with Coomassie blue (bottom), and then transferred to PVDF membranes for Western blotting (top). Arrowheads indicate the positions of the scFv band. M, molecular weight markers.