Platforms for Plant-Based Protein Production
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ABSTRACT:
SUBMITTER: Pavlov A
PROVIDER: S-EPMC7124131 | biostudies-literature | 2018 Jan
REPOSITORIES: biostudies-literature
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Project description:A synthetic inducible operon (IbPSO) expressing alsS, ilvC, ilvD and kivD genes encoding a pathway capable to transform pyruvate into 2-isobutyraldehyde has been designed and two recombinant plasmids named pIZIbPSO and p424IbPSO were constructed. The IbPSO containing plasmids can generate in a single transformation event new recombinant isobutanol producer strains and are useful for testing as suitable hosts wild type bacteria in different culture media. In this way we found that Shimwellia blattae (p424IbPSO) was able to produce in flasks up to 6 g l(-1) of isobutanol using glucose as carbon source. Moreover, for the first time, we have demonstrated that isobutanol can be produced from sucrose using Escherichia coli W (ATCC9367) transformed with pIZIbPSO. These robust recombinant strains were also able to produce isobutanol from a raw carbon source like hydrolysed lignocellulosic biomass.
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Project description:Myoglobin is a heme-protein in the muscle of vertebrates with important functions in the oxygenation of tissues and as a regulator in nitric oxide signaling. Myoglobin from many species is also an important nutritional source of bioavailable iron. In this study, we have successfully produced human myoglobin in the leaves of Nicotiana benthamiana by transient expression using a viral vector delivered by Agrobacterium tumefaciens. Analyses confirmed that heme was incorporated and the protein was functional, with observed properties consistent with those of native myoglobins. A relatively high degree of purity could be achieved with low cost methods. The results show the high potential of plants as a production platform for heme proteins, a group of proteins of interest for iron nutrition applications and possible future pharmaceutical development.
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Project description:Soybean (Glycine max) offers an important source of plant-based protein. Currently much of Europe's soybean is imported, but there are strong economic and agronomic arguments for boosting local production. Soybean is grown in central and eastern Europe but is less favoured in the North due to climate. We conducted field trials across three seasons and two sites in the UK to test the viability of early-maturing soybean varieties and used the data from these trials to calibrate and validate the Rothamsted Landscape Model. Once validated, the model was used to predict the probability soybean would mature and the associated yield for 26 sites across the UK based on weather data under current, near-future (2041-60) and far-future (2081-2100) climate. Two representative concentration pathways, a midrange mitigation scenario (RCP4.5) and a high emission scenario (RCP8.5) were also explored. Our analysis revealed that under current climate early maturing varieties will mature in the south of the UK, but the probability of failure increases with latitude. Of the 26 sites considered, only at one did soybean mature for every realisation. Predicted expected yields ranged between 1.39 t ha-1 and 1.95 t ha-1 across sites. Under climate change these varieties are likely to mature as far north as southern Scotland. With greater levels of CO2, yield is predicted to increase by as much as 0.5 t ha-1 at some sites in the far future, but this is tempered by other effects of climate change meaning that for most sites no meaningful increase in yield is expected. We conclude that soybean is likely to be a viable crop in the UK and for similar climates at similar latitudes in Northern Europe in the future but that for yields to be economically attractive for local markets, varieties must be chosen to align with the growing season.
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Project description:Microgravity-induced bone loss is a main obstacle for long term space missions as it is difficult to maintain bone mass when loading stimuli is reduced. With a typical bone mineral density loss of 1.5% per month of microgravity exposure, the chances for osteoporosis and fractures may endanger astronauts' health. Parathyroid Hormone or PTH (1-34) is an FDA approved treatment for osteoporosis, and may reverse microgravity-induced bone loss. However, PTH proteins requires refrigeration, daily subcutaneous injection, and have a short shelf-life, limiting its use in a resource-limited environment, like space. In this study, PTH was produced in an Fc-fusion form via transient expression in plants, to improve the circulatory half-life which reduces dosing frequency and to simplify purification if needed. Plant-based expression is well-suited for space medicine application given its low resource consumption and short expression timeline. The PTH-Fc accumulation profile in plant was established with a peak expression on day 5 post infiltration of 373 ± 59 mg/kg leaf fresh weight. Once the PTH-Fc was purified, the amino acid sequence and the binding affinity to its target, PTH 1 receptor (PTH1R), was determined utilizing biolayer interferometry (BLI). The binding affinity between PTH-Fc and PTH1R was 2.30 × 10-6 M, similar to the affinity between PTH (1-34) and PTH1R (2.31 × 10-6 M). Its function was also confirmed in a cell-based receptor stimulation assay, where PTH-Fc was able to stimulate the PTH1R producing cyclic adenosine monophosphate (cAMP) with an EC50 of (8.54 ± 0.12) x 10-9 M, comparable to the EC50 from the PTH (1-34) of 1.49 × 10-8 M. These results suggest that plant recombinant PTH-Fc exhibits a similar binding affinity and potency in a PTH1R activation assay compared to PTH. Furthermore, it can be produced rapidly at high levels with minimal resources and reagents, making it ideal for production in low resource environments such as space.
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Project description:Automated high-throughput plant phenotyping (HTPP) enables non-invasive, fast and standardized evaluations of a large number of plants for size, development, and certain physiological variables. Many research groups recognize the potential of HTPP and have made significant investments in HTPP infrastructure, or are considering doing so. To make optimal use of limited resources, it is important to plan and use these facilities prudently and to interpret the results carefully. Here we present a number of points that users should consider before purchasing, building or utilizing such equipment. They relate to (1) the financial and time investment for acquisition, operation, and maintenance, (2) the constraints associated with such machines in terms of flexibility and growth conditions, (3) the pros and cons of frequent non-destructive measurements, (4) the level of information provided by proxy traits, and (5) the utilization of calibration curves. Using data from an Arabidopsis experiment, we demonstrate how diurnal changes in leaf angle can impact plant size estimates from top-view cameras, causing deviations of more than 20% over the day. Growth analysis data from another rosette species showed that there was a curvilinear relationship between total and projected leaf area. Neglecting this curvilinearity resulted in linear calibration curves that, although having a high r2 (> 0.92), also exhibited large relative errors. Another important consideration we discussed is the frequency at which calibration curves need to be generated and whether different treatments, seasons, or genotypes require distinct calibration curves. In conclusion, HTPP systems have become a valuable addition to the toolbox of plant biologists, provided that these systems are tailored to the research questions of interest, and users are aware of both the possible pitfalls and potential involved.
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Project description:Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1?11.000 and 1?39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using ?Pf38 antibodies demonstrated strong inhibition (?60%) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by ?Pf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.
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Project description:Dengue fever is endemic in more than 120 countries, which account for 3.9 billion people at risk of infection worldwide. The absence of a vaccine with effective protection against the four serotypes of this virus makes differential molecular diagnosis the key step for the correct treatment of the disease. Rapid and efficient diagnosis prevents progression to a more severe stage of this disease. Currently, the limiting factor in the manufacture of dengue (DENV) diagnostic kits is the lack of large-scale production of the non-structural 1 (NS1) protein (antigen) to be used in the capture of antibodies from the blood serum of infected patients. In this work, we use plant biotechnology and genetic engineering as tools for the study of protein production for research and commercial purposes. Gene transfer, integration and expression in plants is a valid strategy for obtaining large-scale and low-cost heterologous protein production. The authors produced NS1 protein of the dengue virus serotype 2 (NS1DENV2) in the Arabidopsis thaliana plant. Transgenic plants obtained by genetic transformation expressed the recombinant protein that was purified and characterized for diagnostic use. The yield was 203 μg of the recombinant protein per gram of fresh leaf. By in situ immunolocalization, transgenic protein was observed within the plant tissue, located in aggregates bodies. These antigens showed high sensitivity and specificity to both IgM (84.29% and 91.43%, respectively) and IgG (83.08% and 87.69%, respectively). The study goes a step further to validate the use of plants as a strategy for obtaining large-scale and efficient protein production to be used in dengue virus diagnostic tests.
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Project description:Plant-based systems are safe alternatives to the current platforms for the production of biologically active therapeutic proteins. However, plant-based expression systems face certain major challenges, including the relatively low productivity and the generation of target proteins in biologically active forms. The use of plant virus-based expression systems has been shown to enhance yields, but further improvement is still required to lower the production cost. In this study, various strategies were employed to increase the yields of an important therapeutic protein, human interferon gamma (IFNγ), in Nicotiana benthamiana through modifications of expression vectors based on potexviruses. Among these, the vector based on a coat protein (CP)-deficient Bamboo mosaic virus (BaMV), pKB△CHis, was shown to exhibit the highest expression level for the unmodified IFNγ. Truncation of the N-terminal signal peptide of IFN (designated mIFNγ) resulted in a nearly seven-fold increase in yield. Co-expression of a silencing suppressor protein by replacing the coding sequence of BaMV movement protein with that of P19 led to a 40% increase in mIFNγ accumulation. The fusion of endoplasmic reticulum (ER) retention signal with mIFNγ significantly enhanced the accumulation ratio of biologically active dimeric mIFNγ to 87% relative to the non-active monomeric form. The construct pKB19mIFNγER, employing the combination of all the above enhancement strategies, gave the highest level of protein accumulation, up to 119 ± 0.8 μg/g fresh weight, accounting for 2.5% of total soluble protein (TSP) content. These findings advocate the application of the modified BaMV-based vector as a platform for high-level expression of therapeutic protein in N. benthamiana.
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