709 SummaryEthanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β -1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall.With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T 2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

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.

SummaryThe 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.

The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.