RESUMEN
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.
Asunto(s)
Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Semillas/genética , Zea mays/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ingeniería Genética/métodos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Hibridación de Ácido Nucleico/métodos , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Semillas/embriología , Semillas/metabolismo , Zea mays/embriología , Zea mays/metabolismoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Variación Genética , Proteínas de Choque Térmico/genética , Regiones Promotoras Genéticas/genética , Ubiquitina C/genética , Zea mays/genética , Secuencia de Bases , ADN de Plantas/genética , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.