Localization of a bacterial protein in starch granules of transgenic maize kernels

Abstract

The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with potential for use as a vaccine, a carrier molecule to deliver antigens to gut-associated lymphoid tissues, and possibly an adjuvant to make coadministered vaccines more effective. LT-B produced in plants was shown to be functional and immunogenic in animals and humans. In this work, we show that maize-derived LT-B is strongly associated with starch granules in endosperm. Using immunogold labeling/electron microscopy, cell fractionation, and protein analysis techniques, we observed that LT-B protein could be detected both internally and externally in starch granules. This strong association confers an effective copurification of the antigen with the starch fraction of maize kernels, thermostability desirable in maize processing, and resistance to peptic degradation in simulated gastric fluid digests, an important attribute for an orally delivered antigen.

Fig. 1.

Schematic diagram of constructs P77 and P81 used for maize transformation to generate LT-B-expressing transgenic plants. Both constructs are in a pUC19 vector and contain the 27-kDa maize γ-zein promoter, tobacco etch virus (TEV) translational enhancer leader sequence, sLT-B, and soybean vegetative storage protein terminator (Tvsp). SP, signal peptide. P77, sLT-B fused to the bacterial signal peptide from LT-B; P81, sLT-B fused to signal peptide from the maize 27 kDa γ-zein protein.

Fig. 2.

Immunolocalization of LT-B in immature maize kernels. (A) Section of maize kernel showing a cell beneath the aleurone layer. C, cell wall; N, nucleus; S, starch granules. (B) P77 transgenic kernels showing LT-B localization in starch granules. (C) GBSS localization in starch granules. (D) α-Zein protein localization in cytoplasm. Arrowheads indicate gold particles. (Bars = 500 nm at ×18,000.)

Fig. 3.

Western blot analyses of total proteins from starch samples after thermolysin treatment. The samples were separated on a SDS/12% PAGE, transferred to a 0.45-μm nitrocellulose membrane, and probed with goat anti-LT-B antibodies (A), rabbit anti-zein antibodies (B), or rabbit anti-GBSS (C), followed by rabbit anti-goat or goat anti-rabbit alkaline phosphatase conjugate, respectively. P77 starch samples washed with 75% ethanol plus 3% β-mercaptoethanol (lane 1), 75% ethanol (lane 2), or 95% ethanol (lane 3), and B73 nontransgenic starch samples washed with 75% ethanol plus 3% β-mercaptoethanol (lane 4), respectively. Solid arrows in A, monomeric form of LT-B (11.6 kDa); open arrow in B, zein proteins (19 and 22 kD); arrowhead in C, GBSS (60 kD).

Fig. 4.

Percentage of LT-B in total protein (A) and TAEP (B) of maize kernel fractions. E, endosperm fraction; S, starch fraction.

Fig. 5.

Retention of functional (G_M1-binding) LT-B in TAEP after 4 h of incubation at 37, 55, 65, and 85°C. (A) Ground maize meal. (B) Purified maize starch. Solid lines, transgenic maize line P77; dotted lines, nontransgenic maize control spiked with 1 μg/ml of bacterium-derived soluble LT-B.

Fig. 6.

Western blot analysis of LT-B after gastric fluid digestion simulation. (A1 and A2) Ground maize kernels mixed with bacterial LT-B. (B1 and B2) Ground transgenic maize kernels. The samples were treated with pepsin at various length of period and separated on an SDS/18% PAGE and transferred to a 0.45-μm nitrocellulose membrane and probed with rabbit anti-LT-B antibodies. Lanes 1, 5, 10, and 15, 0-min digestion (no digestion control); lanes 2 and 6, 30-s digestion; lanes 3 and 7, 1-min digestion; lanes 4 and 8, 5-min digestion; lanes 9, 11, and 16, 15-min digestion; lanes 12 and 17, 30-min digestion; lanes 13 and 18, 60-min digestion; lane 14, 120-min digestion.