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Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and

Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction.

Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction. Recombinantly expressed BChE, however, tends to be in monomer or dimer oligomeric forms, which are far less stable than the tetramer form of the enzyme. When BChE is transiently expressed in Nicotiana benthamiana, it is produced mainly as monomers and dimers. However, when the protein is expressed through stable transformation, it produces much greater proportions of tetramers. Tetramerization of WT human plasma derived BChE is facilitated by the binding of a proline rich peptide. In this thesis, I investigated if a putative plant-derived analog of the mammalian proline-rich attachment domain caused stably expressed cocaine hydrolase variants of human BChE to undergo tetramerization. I also examined if co-expression of peptides with known proline-rich attachment domains further shifted the monomer-tetramer ratio toward the tetramer.
ContributorsKendle, Robert Player (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Larrimore, Kathy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high

Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh (Thesis director) / Mor, Tsafrir (Committee member) / Diamos, Andrew (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Over the last century, society has begun to acknowledge and observe how human actions are negatively impacting the environment. Sustainable living is becoming more adopted into daily lives, including a focus on waste management and recycling. Previous informal studies have proposed that coffee grounds can be recycled and added to

Over the last century, society has begun to acknowledge and observe how human actions are negatively impacting the environment. Sustainable living is becoming more adopted into daily lives, including a focus on waste management and recycling. Previous informal studies have proposed that coffee grounds can be recycled and added to the soil to increase plant productivity. The objective of this experiment was to test how different concentrations of roasted coffee grounds would affect the overall plant productivity when introduced in the soil of various plant types and environmental atmospheres. Three treatments were selected (100% potting mix, 50% potting mix/50% coffee grounds, and 25% potting mix/75% coffee grounds) and applied to 3 acid-tolerating plants (radish, basil, and parsley). Each of these treatments were grown in 2 different environments, where one was planted in a Tempe, AZ backyard while the other group was planted in a lab environment, locating at Arizona State University's Tempe Campus. Each plant with its respective treatments (plant type, coffee ground treatment, and environment) had 10 identical plants for statistical accuracy, resulting in a total of 180 plants grown, observed, and analyzed for this 3-month long experiment. The plant development, plant height, length of roots, quantity of leaves, and environmental observations were recorded and used to define plant productivity in this investigation. The experiment demonstrated low survival rates in all groups including the control group, suggesting a flaw in the experimental design. Nonetheless, the experiment showed that among the surviving plants, the 75% treatment had the largest negative impact on plant productivity. The measured root lengths and leaf quantity had various results across each plant group, leaving the hypothesis unverified. Overall, the experiment was effective in demonstrating negative impacts of great concentrations of coffee grounds when introduced to various plants, but further investigation with an adjusted experimental design will need to be completed to reach a reliable conclusion.
ContributorsVan Winkle, Delaney Dare (Author) / Bang, Christofer (Thesis director) / Fox, Peter (Committee member) / Earl, Stevan (Committee member) / School of Sustainability (Contributor) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description
Plants are essential to human life. They release oxygen into the atmosphere for us to breathe. They also provide shelter, medicine, clothing, tools, and food. For many people, the food that is on their tables and in their supermarkets isn't given much thought. Where did it come from? What part

Plants are essential to human life. They release oxygen into the atmosphere for us to breathe. They also provide shelter, medicine, clothing, tools, and food. For many people, the food that is on their tables and in their supermarkets isn't given much thought. Where did it come from? What part of the plant is it? How does it relate to others in the plant kingdom? How do other cultures use this plant? The most many of us know about them is that they are at the supermarket when we need them for dinner (Nabhan, 2009) (Vileisis, 2008).
ContributorsBarron, Kara (Author) / Landrum, Leslie (Thesis director) / Swanson, Tod (Committee member) / Pigg, Kathleen (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
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Description

As the world’s population exponentially grows, more food production is required. This increasing food production currently has led to the un-sustainable production of chemical fertilizers and resultant overuse. A more sustainable option to enhance food production could be the use of fertilizer derived from food waste. To address this, we

As the world’s population exponentially grows, more food production is required. This increasing food production currently has led to the un-sustainable production of chemical fertilizers and resultant overuse. A more sustainable option to enhance food production could be the use of fertilizer derived from food waste. To address this, we investigated the possibility of utilizing a fertilizer derived from food waste to grow hydroponic vegetables. Arugula (Eruca sativa) ‘Slow Bolt’ and lettuce (Lactuca sativa) ‘Cherokee’ and ‘Rex’ were cultivated using indoor deep-flow hydroponic systems at 23 ºC under a photosynthetic photon flux density of 170 µmol∙m−2∙s−1 with an 18-hour photoperiod. Plant nutrient solutions were provided by food waste fertilizer or commercial 15:5:20 NPK fertilizer at the identical electrical conductivity (EC) of 2.3 mS·cm–1. At the EC of 2.3 mS·cm–1, chemical fertilizer contained 150 ppm N, 50 ppm P, and 200 ppm K, while food waste fertilizer had 60 ppm N, 26 ppm P, and 119 ppm K. Four weeks after the nutrient treatments were implemented, compared to plants grown with chemical fertilizer, lettuce ‘Rex’ grown with food waste fertilizer had four less leaves, 27.1% shorter leaves, 68.2% and 23.1% less shoot and root fresh weight, respectively. Lettuce ‘Cherokee’ and arugula grown with food waste fertilizer followed a similar trend with fresh shoot weights that were 80.1% and 95.6% less compared to the chemical fertilizer, respectively. In general, the magnitude of reduction in the plant growth was greatest in arugula. These results suggest that both fertilizers were able to successfully grow lettuce and arugula, although the reduced plant growth with the food waste fertilizer in our study is likely from a lower concentration of nutrients when we considered EC as an indicator of nutrient concentration equivalency of the two fertilizer types.

ContributorsCherry, Hannah Nichole (Author) / Park, Yujin (Thesis director) / Penton, Ryan (Committee member) / Chen, Zhihao (Committee member) / Environmental and Resource Management (Contributor, Contributor) / College of Integrative Sciences and Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description
Plant viral vectors have previously been used to produce high expression levels of antibodies and other proteins of interest. By utilizing a transformed Agrobacterium with the vector containing the protein of interest for infiltration, viral vectors can easily reach the plant cells making it an effective form of transient protein

Plant viral vectors have previously been used to produce high expression levels of antibodies and other proteins of interest. By utilizing a transformed Agrobacterium with the vector containing the protein of interest for infiltration, viral vectors can easily reach the plant cells making it an effective form of transient protein expression. For this project two different plant viral vectors were compared; the geminiviral vector derived from Bean yellow dwarf virus (BeYDV) and the MagnICON vector system derived from Tobacco Mosaic Virus(TMV) and Potato Virus X(PVX). E16, an antibody against West Nile virus, has previously been expressed using both systems but expression levels between the systems were not directly compared. Agrobacterium tumefaciens EHA105 cells were transformed with both systems and expression levels of E16 were quantified using ELISAs. Results showed very low expression levels of E16 using the geminiviral vector indicating a need for further investigation into the clone used as previous studies reported much higher expression levels with the system.
ContributorsMurphy, Skylar (Author) / Chen, Qiang (Thesis director) / Jugler, Collin (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description
HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to

HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to be produced. The HIV-1 envelope glycoprotein41 and the Gag structural protein have been identified to be particularly important in HIV-1 transcytosis and cytotoxic lymphocyte response, respectively. Enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of glycoprotein (dgp41) comprising the membrane proximal external region (MPER), transmembrane domain and cytoplasmic tail may present a unique and safe way of presenting these proteins in a state mimicking their natural formation. Another form of presenting the immunogenic glycoprotein41, particularly the MPER component, is by presenting it onto the N-terminal of an IgG molecule, thereby creating an IgG fusion molecule. In our lab, both VLPs and IgG fusion molecules are highly expressed and purified within GnGn Nicotiana benthamiana. The results indicated that these recombinant proteins can be assembled properly within plants and can elicit an immune response in mice. This provides a preliminary step in using such Gag/dpg41 VLPs and RIC as present a safe, effective, and inexpensive HIV vaccine.
ContributorsGarcia, Izamar (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05