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.

Criteria for high-level expression of a fungal laccase gene in transgenic maize.

Expression of industrial enzymes in transgenic plants offers an alternative system to fungal fermentation for large-scale production. Very high levels of expression are required to make the enzymes cost-effective. We tested several parameters to determine the best method for achieving high levels of expression for a fungal laccase gene. Transgenic maize plants were generated using an Agrobacterium-mediated system. The molecular parameters that induced the highest expression were the maize embryo-preferred globulin 1 promoter and targeting of the protein to the cell wall. Two independent transgenic events that yielded multiple clonal plants were characterized in detail. Independent transgenic events 01 and 03 contained two or one copies of T-DNA, respectively. Plants derived from a single transgenic event varied in expression level, and the variation in expression levels was heritable. Within the seed, expression in these plants was primarily within the embryo, and was associated with seed browning and limited germination. High oil germplasm was used to increase germination, as well as to assist in increasing expression 20-fold in five generations through breeding and selection.

Advantageous features of plant-based systems for the development of HIV vaccines.

Plants have recently become an attractive option for the production of recombinant proteins. Plant-based systems can be used to produce many classes of foreign proteins including candidate vaccine antigens. The selected antigen can be purified from plant material prior to delivery by the preferred route, or alternatively delivered orally in edible plant material that has been processed to give a homogeneous and stable product. Several plant species have been used to express a wide range of vaccine candidates with tobacco, potato and corn being particularly favored. Corn seed is especially well suited to various food processing technologies that generate dry homogeneous material suitable for extended storage and refrigeration-free transport and distribution. Many antigens have been expressed in corn and assessed for efficacy in trials with generally positive results. Candidate HIV vaccines are particularly good targets for plant-based oral delivery since there is a great need for an easily distributed affordable vaccine that could be administered without injection and induce strong mucosal immune responses. As a first step in evaluating plant expression technology with a relevant antigen that might easily be tested in an animal system, we expressed the SIV major surface glycoprotein gp130 (analogous to HIV gp120) in corn seed. Expression levels were achieved that are compatible with conducting oral delivery trials in animals.