Expression of the sweet protein brazzein in maize for production of a new commercial sweetener.

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.

Delivery of subunit vaccines in maize seed.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.

Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues.

Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2,2,7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7)GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G- and m(3)(2,2,7)G-containing caps (K(as) 2 x 10(6) M(-1) to 7 x 10(6) M(-1)) whereas IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K(as) on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.

Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen.

Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.

Protection of cap-dependent protein synthesis in vivo and in vitro with an eIF4G-1 variant highly resistant to cleavage by Coxsackievirus 2A protease.

The shutoff of host protein synthesis by certain picornaviruses is mediated, at least in part, by proteolytic cleavage of eIF4G-1. Previously, we developed a cleavage site variant of eIF4G-1, termed eIF4G-1(SM), that was 100-fold more resistant to in vitro cleavage by Coxsackievirus 2A protease (2A(Pro)) than wild-type eIF4G-1 (eIF4G-1(WT)), but it was still digested at high protease concentrations. Here we identified a secondary cleavage site upstream of the primary site. We changed Gly at the P1'-position of the secondary site to Ala to produce eIF4G-1(DM). eIF4G-1(DM) was 1,000-10,000-fold more resistant to cleavage in vitro than eIF4G-1(WT). Full functional activity of eIF4G-1(DM) was demonstrated in vitro by its ability to restore cap-dependent translation to a 2A(Pro)-pretreated rabbit reticulocyte system. An isoform containing the binding site for poly(A)-binding protein, eIF4G-1e(DM), was more active in this assay than an isoform lacking it, eIF4G-1a(DM), but only with polyadenylated mRNA. Functional activity was also demonstrated in vivo with stably transfected HeLa cells expressing eIF4G-1(DM) from a tetracycline-regulated promoter. Cap-dependent green fluorescent protein synthesis was drastically inhibited by 2A(Pro) expression, but synthesis was almost fully restored by induction of either eIF4G-1a(DM) or eIF4G-1e(DM). By contrast, encephalomyocarditis virus internal ribosome entry site-dependent green fluorescent protein synthesis was stimulated by 2A(Pro); stimulation was suppressed by eIF4G-1e(DM) but not eIF4G-1a(DM).

A corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine.

Recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. Cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. A subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. Here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals' serum, colostrum and milk. Thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization.

Corn as a production system for human and animal vaccines.

The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery. Here, we demonstrate that the Lt-B protein of enterotoxigenic E. coli is evenly distributed in defatted corn germ prepared from transgenic grain. Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.