Plastid transformation as an expression tool for plant-derived biopharmaceuticals.
ABSTRACT The production of biopharmaceuticals in plants is currently one of the most attractive approaches to modern medicine. Several efficient plant-based expression systems have been developed so far. Among them, plastid transformation has attracted biotechnologists because the plastid genome, unlike nuclear genome, bears a number of unique advantages for plant genetic engineering. These include higher levels of protein production, uniform gene expression of transformants due to the lack of epigenetic interference, and expression of multiple genes (as in operons) from the same construct. Further, the plastid transformation technology is an environmentally friendly method because plastid and their genetic information are maternally inherited in many species with a consequent lack of transmission of plastid DNA by pollen. Recently, great progress has been made with plastid-based production of biopharmaceuticals demonstrating that it is a promising platform for such purposes. This chapter describes detailed protocols for plastid transformation including the delivery of DNA by biolistic method, the selection/regeneration of transplastomic plants, and the molecular analyses to select homoplasmic plants and confirm transgene expression.
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ABSTRACT: The main strategy for resistance to the herbicide glyphosate in plants is the overexpression of an herbicide insensitive, bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance strategy based on the ability to degrade the herbicide can be useful to reduce glyphosate phytotoxicity to the crops. Here we present the characterization of glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved in vitro by a protein engineering approach to efficiently degrade glyphosate. Two constructs were used, one with (GO(TP+)) and one without (GO(TP-)) the pea rbcS plastid transit peptide. Molecular and biochemical analyses confirmed the stable integration of the transgene and the correct localization of the plastid-imported GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both in vitro and in vivo. Two GO(TP+) lines showed moderate resistance to the herbicide in both conditions. Optimization of expression of this GO variant may allow to attain sufficient field resistance to glyphosate herbicides, thus providing a resistance strategy based on herbicide degradation.Journal of biotechnology. 06/2014; 184(201):208.
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ABSTRACT: The evolution in proteomics approaches is notable, including quantitative proteomics and strategies for elucidation of post-translational modifications. Faster and more accurate mass spectrometers as well as cleverer bioinformatics tolls are making the difference in such advancement. Among the wide range of research in plant proteomics, biopharmaceutical production using plants as "biofactories" and the screening of new activities of new molecules, in this case, peptides, are quite important regarding translational proteomics. The present review is focused on "recombinant proteins and bioactive peptides", with biopharmaceuticals and cyclotides chosen as examples. Their application and challenges are focused on a "translational proteomics" point of view, in order to exemplify some new areas of research based on proteomics strategies. This article is part of a Special Issue entitled: Translational Plant Proteomics.Journal of proteomics 06/2013; · 5.07 Impact Factor