FIG 8.

Relationship between stabilities of guest proteins and growth inhibition of S. cerevisiae. (A) APH sandwich fusions containing Im7 guest proteins with different stabilities were cloned behind the GAL promoter, and cells were grown using 2% galactose. Twenty thousand fully induced yeast cells were inoculated into 100 μl YPD containing increasing concentrations of the antibiotic G418 and incubated at 30°C for 20 h. The growth of S. cerevisiae at 400 μg/ml G418 was plotted against the in vitro stabilities of the Im7 variants. From top to bottom, the strains were AYC29, AYC34, AYC35, AYC39, AYC30, and AYC42, carrying the WT and Im7 variants V33E, L34A, I54V, F15A, and F84A, respectively. (B and C) Western blots quantifying sandwich fusion material obtained from soluble fractions (B) and whole-cell lysates (C). Lanes 1 to 5, strains AYC29 (lanes 1), AYC34 (lanes 2), AYC35 (lanes 3), AYC30 (lanes 4), and AYC42 (lanes 5), with WT Im7 and Im7 variants V33E, L34A, F15A, and F84A at position 55 in APH.