The production of recombinant pharmaceutical proteins in plants

Imagine a world in which any protein, either naturally occurring or designed by man, could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight. This could one day become reality as we learn to harness the power of plants for the production of recombinant proteins on an agricultural scale. Molecular farming in plants has already proven to be a successful way of producing a range of technical proteins. The first plant-derived recombinant pharmaceutical proteins are now approaching commercial approval, and many more are expected to follow.     

Programming desiccation-tolerance: from plants to seeds to resurrection plants

Desiccation-tolerance (DT) evolved as the key solution to survival on land by the early algal ancestors of terrestrial plants. This 'first' DT involved utilizing rapidly mobilisable repair mechanisms and is still found today in mosses, such as Tortula ruralis, and ferns, such as Mohria caffrorum. The first seed plants lost vegetative DT while investing their seeds with tolerance mechanisms improving their survival in unfavourable environments. The mechanisms of DT in seeds are strongly connected to their developmentally regulated maturation programs. We propose that angiosperm resurrection plants acquired tolerance by re-activating their innate DT mechanisms in their vegetative tissues. Here we review the current hypotheses regarding the genetic evidence for the evolution of DT in resurrection plants. We also present strong evidence showing the activation of seed specific genetic elements in the vegetative tissues of resurrection plants.     

Lectin-associated proteins from the seeds of Leguminosae

The seeds of Pisum sativum (pea), Canavalia ensiformis, Vicia faba, Vicia sativa, and Ricinus communis were shown to contain proteins which are associated to the respective lectins (lectin binders). The lectin binders from Pisum sativum and Canavalia ensiformis were studied more closely. Both are single proteins not resembling the variety of membrane glycoproteins found in animals and plants which bind to lectins. The pea lectin binder is a tetrameric glycoprotein composed of identical subunits of the Mr 51 000. Its interaction with the lectin is abolished by acidic buffers or by glucose. The Concanavalin A binder, which does not contain sugar, is composed of one kind of subunit, Mr of 35 000. As in the case of the pea lectin binder, glucose and acid dissociate the lectin-lectin binder complex, but in contrast to the pea lectin binder low NaCl concentrations also cause this effect. During germination and growth, the Concanavalin A binder appears in the roots.     

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

Seven trypsin inhibitors were isolated from the seeds of Cucurbitaceae plants: two from cucumber (Cucumis sativus) and red bryony (Bryonia diotica) and one from figleaf gourd (Cucurbita ficifolia), spaghetti squash (Cucurbita pepo var. (vegetable spaghetti) and water melon (Citrullus vulgaris). The inhibitors were purified by fractionation with ammonium sulphate, followed by ion-exchange chromatography and affinity chromatography using immobilized trypsin or anhydro-trypsin. The homogeneous inhibitors from cucumber and water melon are made up of 32 and 30 amino acid residues, respectively, whereas the remaining ones of 29 residues. All inhibitors contain three disulphide bridges and are free of threonine, phenylalanine and tryptophan. Inhibitors from spaghetti squash and CSTI IIb from cucumber are inactivated by acetylation of free amino groups whereas the remaining ones are inactivated by modification of arginine with 1,2-cyclohexanedione. Thus the P1 residues of the reactive sites of the inhibitors are lysine and arginine, respectively.     

Enzyme inhibitors from plants: Enterokinase inhibitors in tubers and seeds

Of the 22 tubers and 9 pulses screened for inhibitors of enterokinase activity, the following 12 tubers,Curcuma amada, Kyllinga monocephala, Solanum tuberosum, Canna indica, Helianthus tuberosus, Coleus parviformis, Mirabilis jalapa, Colocasia antiquorum (red variety),Alium cepa, Amorphophalus companulatus, Maranta arundinacea, Daucus carota, and 9 pulses namely,Vigna sinensis, Arachis hypogea, Pisum sativum, Phaseolus vulgaris (white bean),Phaseolus vulgaris (kidney bean),Phaseolus mungo, Cicer arietinum, Dolichos lablab and Cajonus cajan contained inhibitory activity. Three tubers,Amorphophalus companulatus, Maranta arundinacea andDaucus carota and all the nine pulses exhibited endogenous esterase activity towards benzoyl arginine ethyl ester. Among the 8 pulses and 3 tubers processed by affinity chromatography on trypsin-sepharose, to separate trypsin inhibitor from enterokinase inhibitor,Phaseolus vulgaris (kidney bean),Phaseolus vulgaris (white bean) andDolichos lablab contained distinct enterokinase inhibitors. These fractions were devoid of trypsin inhibitor activity. The trypsin inhibitor fromColeus parviformis tubers alone did not bind to trypsinsepharose and was recovered in the unbound fraction along with the enterokinase inhibitor.     

Investigating detection success: lessons from trials using decoy rare plants

Imperfect detection leads to underestimates of species presence and decreases the reliability of survey data. Imperfect detection has not been examined in detail for boreal forest understory plants, despite widespread use of surveys for rare plants prior to development. We addressed this issue using detectability trials conducted in Alberta, Canada with decoy vascular plants. Volunteer observers searched in survey plots for species while unaware of their true presence or abundance. Our findings indicate that the detection of cryptic species is very low when abundance is low (0–35%) and plot size is large (<?50% in?≥?100 m²). Plant density (individuals per unit area) was the most important determinant of detection probability, where more abundant species were detected more often and with less survey effort. When abundance was held constant, diffusely arranged species were twice as likely to be detected compared to those in clumps. Detection of cryptic species can be low even when individuals are flowering, and even morphologically distinct species can go unnoticed in small plots. We suggest that future decoy trials investigate search strategies that could improve detection and that field surveys for vascular plants address imperfect detection through careful consideration of plot size, characteristics of the target species, and survey effort, both in terms of time expenditure within an area and the number of observers employed.     

Sowing the seeds for a career in medicine: reflections and projections

Dr. George Lister delivered the following presentation as the Lee E. Farr Lecturer on May 8, 2011, which served as the culmination of the annual Student Research Day at Yale School of Medicine. He is the Chair of Pediatrics at the University of Texas Southwestern Medical School and Pediatrician-in-Chief at Children's Medical Center of Dallas. In his lecture to the medical students, who had just completed their research theses, Dr. Lister discusses his own work on sudden infant death syndrome (SIDS), demonstrating the complexity of clinical research and proving insight into the traits required of physician scientists. Committed to medical education and recognized by several awards for his mentorship, he ends the talk by imparting valuable advice on future physicians.     

Storage proteins from seeds of Pinus pinea L

The Mediterranean stone pine Pinus pinea L. (gymnosperm, Pinaceae) is much appreciated for its seed production, widely used in food preparation in the Mediterranean Basin. Seeds contain 25% proteins on a dry-weight basis. Pinus pinea accumulate globulins as major storage proteins in seeds (75% of total storage proteins), composed of several subunits of 10 to 150 kDa, revealed by SDS-PAGE. The albumin fraction (15%) represents three subunits of 14, 24 and 46 kDa. Glutelins, the least soluble fraction, represents a small proportion (10%). Their constitutive units have frequent PM of 43 kDa. Prolamins also represent a very small percentage (1 to 2%).     

Seed use in the field: delivering seeds for restoration success

Seed delivery to site is a critical step in seed‐based restoration programs. Months or years of seed collection, conditioning, storage, and cultivation can be wasted if seeding operations are not carefully planned, well executed, and draw upon best available knowledge and experience. Although diverse restoration scenarios present different challenges and require different approaches, there are common elements that apply to most ecosystems and regions. A seeding plan sets the timeline and details all operations from site treatments through seed delivery and subsequent monitoring. The plan draws on site evaluation data (e.g. topography, hydrology, climate, soil types, weed pressure, reference site characteristics), the ecology and biology of the seed mix components (e.g. germination requirements, seed morphology) and seed quality information (e.g. seed purity, viability, and dormancy). Plan elements include: (1) Site treatments and seedbed preparation to remove undesirable vegetation, including sources in the soil seed bank; change hydrology and soil properties (e.g. stability, water holding capacity, nutrient status); and create favorable conditions for seed germination and establishment. (2) Seeding requirements to prepare seeds for sowing and determine appropriate seeding dates and rates. (3) Seed delivery techniques and equipment for precision seed delivery, including placement of seeds in germination‐promotive microsites at the optimal season for germination and establishment. (4) A monitoring program and adaptive management to document initial emergence, seedling establishment, and plant community development and conduct additional sowing or adaptive management interventions, if warranted. (5) Communication of results to inform future seeding decisions and share knowledge for seed‐based ecological restoration.     

Circular proteins from plants and fungi

Circular proteins, defined as head-to-tail cyclized polypeptides originating from ribosomal synthesis, represent a novel class of natural products attracting increasing interest. From a scientific point of view, these compounds raise questions of where and why they occur in nature and how they are formed. From a rational point of view, these proteins and their structural concept may be exploited for crop protection and novel pharmaceuticals. Here, we review the current knowledge of three protein families: cyclotides and circular sunflower trypsin inhibitors from the kingdom of plants and the Amanita toxins from fungi. A particular emphasis is placed on their biological origin, structure, and activity. In addition, the opportunity for discovery of novel circular proteins and recent insights into their mechanism of action are discussed.     

Estimating the invasion success of introduced plants

We present methods for estimating the base proportion of introduced alien species that will naturalize, and the distribution of time until naturalization occurs. The approach is Bayesian, incorporating prior estimates of the probability of naturalization and the time from introduction to naturalization. A worked example uses data on the introduction and time to recorded naturalization of woody perennials introduced to South Australia. Up until 2007, 188 of 2230 (8.4%) woody perennials listed in nursery catalogues between 1843 and 1985 were recorded as having naturalized. If prior information on naturalization rates from elsewhere is ignored, the available data suggest that the most likely proportion of introduced plants that will naturalize is large (0.93) though uncertain (95% CI 0.51–0.99), with the corresponding mean time to recorded naturalization being protracted (522 years) and similarly uncertain (95% CI 360–678 years). Alternatively, if informative prior estimates of both the naturalization probability and the time to recorded naturalization are incorporated, the most likely probability of naturalization is estimated to be 18.6% (95% CI 15.5–23.4%). For those plants that do naturalize, the most likely value for the mean time from importation to recorded naturalization is 149 years (95% CI 130–174 years). Our results illustrate the potentially long timescale of the naturalization process, and the challenges this presents for obtaining accurate estimates of naturalization parameters.     

Considerations for the recovery of recombinant proteins from plants

The past 5 years have seen the commercialization of two recombinant protein products from transgenic plants, and many recombinant therapeutic proteins produced in plants are currently undergoing development. The emergence of plants as an alternative production host has brought new challenges and opportunities to downstream processing efforts. Plant hosts contain a unique set of matrix contaminants (proteins, oils, phenolic compounds, etc.) that must be removed during purification of the target protein. Furthermore, plant solids, which require early removal after extraction, are generally in higher concentration, wider in size range, and denser than traditional bacterial and mammalian cell culture debris. At the same time, there remains the desire to incorporate highly selective and integrative separation technologies (those capable of performing multiple tasks) during the purification process from plant material. The general plant processing and purification scheme consists of isolation of the plant tissue containing the recombinant protein, fractionation of the tissue along with particle size reduction, extraction of the target protein into an aqueous medium, clarification of the crude extract, and finally purification of the product. Each of these areas will be discussed here, focusing on what has been learned and where potential concerns remain. We also present details of how the choice of plant host, along with location within the plant for targeting the recombinant protein, can play an important role in the ultimate ease of recovery and the emergence of regulations governing plant hosts. Major emphasis is placed on three crops, canola, corn, and soy, with brief discussions of tobacco and rice.