Figure 3.

Lyophilization and characterization. (a) Amount of CTB-EX4 protein in fresh (F) and lyophilized (L) leaves. (b) Western blot analysis of fresh and lyophilized leaves expressing CTB-EX4. Equal quantity (50 mg) of fresh and lyophilized material was extracted in same volume (300 μl) of extraction buffer. Samples were loaded in a serial dilution as indicated. Western blot analysis with anti-CTB (c) and anti-EX4 (d) polyclonal antibody to evaluate long-term stability of lyophilized CTB-EX4 after storage at room temperature for 10 months. F, fresh leaf; L, 10-month old lyophilized leaf. Total leaf protein (5 μg) was loaded in each lane. CTB, Positive or negative control (20 ng). Samples were incubated for 10 min with DTT (100 mM) or boiled for 2 minutes. (e) Western blot analysis to evaluate stability of PA in lyophilized lettuce leaves after storage at room temperature for 2 (1), 4 (2), and 6 (3) months. PA, standard (100 ng), WT, untransformed lettuce. Total soluble protein (3 μg) was loaded in each lane. (f) Antigen stability after 3 months of storage, and lyophilization for different durations: (1) 24, (2) 48 and (3) 72 hrs. Fold increase of total protein (g) and specific antigen (h) after lyophilization. PA, lettuce transplastomic plant expressing PA; CTB-Pins, lettuce transplastomic plant expressing CTB-Proinsulin; white bar, fresh material; grey bar, lyophilized material. (i) Western blot analysis of fresh (F) and lyophilized (L) leaves expressing PA. Total soluble protein (10 μl) was loaded after equal quantity (50 mg) was extracted in same volume (300 μl) of extraction buffer. (j) Microbial burden of leaves expressing PA. 1, fresh leaf; 2, lyophilized leaf; 3, commercially available freeze-dried alfalfa capsules. (k) Simplified diagram of capsulation of lyophilized transplastomic leaf material. Data shown are means ± SD of three independent experiments.