Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.

The production of recombinant proteins in transgenic barley grains

The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3–1, 4)-β-glucanase with the D hordein gene (Hor3–1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an α-amylase promoter and the code for the export signal peptide yielded ≈1 μg⋅mg−1 soluble protein, whereas 54 μg⋅mg−1 soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3–1, 4)-β-glucanase under the control of the Hor3–1 promoter.

Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Increased plant volatile production affects oviposition, but not larval development, in the moth Helicoverpa armigera

It is well established that herbivorous insects respond to changes in plant odour production, but little attention has been given to whether these responses relate to direct fitness costs of plant volatile production on insect growth and survival. Here, we use transgenic Nicotiana tabacum (tobacco) plants that produce relatively large amounts of the volatile (S)-linalool to study whether the responses of egg-laying herbivorous insects to linalool production relate directly to the growth and survival of offspring. In choice tests, fewer eggs were laid on transgenic plants compared with non-transformed controls, indicating that increased linalool emissions have a deterrent effect on Helicoverpa armigera oviposition. Larval survival and larval mass after feeding on transgenic leaves, however, was comparable to non-transformed controls. (S)-linalool, whether in volatile or sequestered form, does not appear to have a direct effect on offspring fitness in this moth. We discuss how the ecology of this polyphagous moth species may necessitate a high tolerance for certain volatiles and their related non-volatile compounds, and suggest that responses by adult female H. armigera moths towards increased linalool production may be context specific and relate to other indirect effects on fitness.

Characterization and Subcellular Compartmentation of Recombinant 4-Hydroxyphenylpyruvate Dioxygenase from Arabidopsis in Transgenic Tobacco1

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Broad resistance to plant viruses in transgenic plants conferred by antisense inhibition of a host gene essential in S-adenosylmethionine-dependent transmethylation reactions.

S-Adenosylhomocysteine hydrolase (SAHH) is a key enzyme in transmethylation reactions that use S-adenosylmethionine as the methyl donor. Because of the importance of SAHH in a number of S-adenosylmethionine-dependent transmethylation reactions, particularly the 5' capping of mRNA during viral replication, SAHH has been considered as a target of potential antiviral agents against animal viruses. To test the possibility of engineering a broad type of resistance to plant viruses, we expressed the antisense RNA for tobacco SAHH in transgenic tobacco plants. As expected, transgenic plants constitutively expressing an anti-sense SAHH gene showed resistance to infection by various plant viruses. Among those plants, about half exhibited some level of morphological change (typically stunting). Analysis of the physiological change in those plants showed that they contained excess levels of cytokinin. Because cytokinin has been found to induce acquired resistance, there is also a strong possibility that the observed resistance was induced by cytokinin.

Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

Plant-based immunocontraceptive control of wildlife - "potentials, limitations, and possums"

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immuno-contraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests. (c) 2004 Elsevier Ltd. All rights reserved.

Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

Third International Conference on plant-based vaccines and antibodies

This relatively new biennial meeting - the first was in Prague in 2005 - was chaired by Julian Ma (Guy's Hospital, London, UK), with Mario Pezzotti (University of Verona, Italy) as local organizer, and attracted approximately 180 delegates from 25 countries. The theme was 'Plant Expression Systems for Recombinant Pharmacologics': there were 46 talks gathered into two plenaries, 12 themed sessions and 72 posters. Topics covered included publicly funded and commercial developments, innovation, regulation and commercialization, competition with conventional technology, manufacture and new products. © 2009 Expert Reviews Ltd.

Riding the tide of biopharming in Africa: Considerations for risk assessment

In the past few years, plant biotechnology has gone beyond traditional agricultural production of food, feed and fibre, and moved to address more complex contemporary health, social and industrial challenges. The new era involves production of novel pharmaceutical products, speciality and fine chemicals, phytoremediation and production of renewable energy resources to replace non-renewable fossil fuels. Plants have been shown to provide a genuine and low-cost alternative production system for high-value products. Currently, the principal plant-made products include antibodies, feed additives, vaccine antigens and hormones for human and animal health, and industrial proteins. Despite the unique advantages of scalability, cost and product safety, issues of politics, environmental impact, regulation and socioeconomics still limit the adoption of biopharmaceuticals, especially in the developing world. Plant-based production systems have further complicated biosafety, gene flow and environmental impact assessments with generally genetically modified plants, topics that are already partially understood. This article provides a background to biopharming, highlighting basic considerations for risk assessment and regulation in developing countries, with an emphasis on plant-based vaccine production in South Africa.

Pilot plant for production of fish ensilage and the economics of production

This communication describes the design aspect and functions of individual pieces of equipment of a pilot plant for the production of fish ensilage based on lactic acid fermentation process. Details about the equipment, process flow sheet and equipment layout of the pilot plant have been given. An attempt has been made to prepare an estimate of the cost of production of liquid ensilage and solid feed mix.