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.

Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production.

The production of heterologous proteins in plants at levels consistent with commercialization of protein products requires molecular tools to ensure high-level transgene expression. The identification of strong promoters, preferably specific to the target expression tissue, is a focus for improving foreign protein yields using transgenic cereals as a production system. Thus, there is a requirement for strong embryo preferred monocot promoters. We obtained the sequences of 500 randomly selected maize cDNA clones to determine gene expression profiles in embryo tissues at multiple stages during development. Promoters corresponding to the most abundant clones were identified and isolated. These promoters were fused to the b-glucuronidase reporter and their tissue specificity and developmental expression characteristics assessed in transgenic maize. All of the isolated promoters tested drove transgene expression predominantly in the embryo and were most active late in embryogenesis during storage protein deposition. One of the most active promoters assessed by transgene expression was associated with the globulin-1 protein. Sequence identified here extended approximately 1.6 kb distal to the previously identified extent of the globulin-1 promoter, and this additional sequence boosted expression over two-fold. The extended globulin-1 promoter sequence isolated in this study has the potential for driving transgene expression at higher levels than those previously reported for cereals. Also, other highly active embryo promoters identified here offer opportunities to express multiple foreign proteins simultaneously at high levels in embryo tissues, while avoiding concerns over gene silencing due to the repeated use of a single promoter.

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.

Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants.

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.