Figure 5

VP1:VP3 optimization with “low glucose-high galactose” induction strategy. (A) After over-night growth on glucose, YEplacRepCap + pYESVP1KM (RepCap + VP1KM) and YEplacp40Cap + pYESVP1KM (Cap + VP1KM) yeast clones were induced in the presence of high glucose (1.5%) and high galactose (2.5%) concentration. VP expression was analyzed by Western blot at three different time points before induction (“0 h”) and after 9 h and 18 h. There was no significant difference in VP1/VP3 expression pattern between clones and the best ratio (1:9), was detected for 9 h induction time for yeast cells co-transformed with YEplacRepCap and pYESVP1KM (RepCap + VP1KM) . (B) After overnight growth on glucose, YEplacRepCap and pYESVP1KM (RepCap + VP1KM) co-transformed yeast cells were induced in the medium containing low glucose (0.5%) and high galactose (5%) concentration. Lanes 0–5: VP1-VP3 expression pattern was determined by Western blot analysis before induction (lane1,“0 h”) and after 5 different induction periods (lane 2, 4.5 h; lane 3, 6 h; lane 4, 7 h; lane 5, 8 h, lane 6, 9 h) . VP1:VP3 ratios, calculated by means of band densitometry, are presented in the table below. Numbers represent the density expressed in arbitrary unit detected by the analysis software described in materials and methods. Results are reported as mean of at least three independent experiment ± standard error. The best ratio was obtained after 4.5 h induction in 0.5% glucose + 5% galactose medium (lane 2).