Matching Items (32)
![153167-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-08/153167-Thumbnail%20Image.png?versionId=B756fl1ziOPHQiTGVb41rJvkjff2PYWA&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T054922Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=864651a6d5e1ec3784a50f88e3ec8cad75bbb814d811fad9b771a8c4e1597b26&itok=X1rYod4f)
Description
The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
ContributorsGong, Zhen (Author) / Fromme, Petra (Thesis advisor) / Mor, Tsafrir (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014
![150811-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-08/150811-Thumbnail%20Image.png?versionId=sGPT23ghdHXWa.IUi_KOCsBkVoQYbRCk&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T071058Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=d69aeea2f65fdcbbc6c2f4aeb4dfb1610bee5ef8ca96bcb1889d19b057347e90&itok=r12AP01r)
Description
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
![154006-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-08/154006-Thumbnail%20Image.png?versionId=AfI5XjmAApOzS0rQLO2NbVOaMHfoB1XO&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T144744Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=0246bd764fe2608a425f3fd2ec6482bd7f31e03fb16bc122a3687bb074337dcc&itok=gjxM_kQA)
Description
Molecular docking serves as an important tool in modeling protein-ligand interactions. Most of the docking approaches treat the protein receptor as rigid and move the ligand in the binding pocket through an energy minimization, which is an incorrect approach as proteins are flexible and undergo conformational changes upon ligand binding. However, modeling receptor backbone flexibility in docking is challenging and computationally expensive due to the large conformational space that needs to be sampled.
A novel flexible docking approach called BP-Dock (Backbone Perturbation docking) was developed to overcome this challenge. BP-Dock integrates both backbone and side chain conformational changes of a protein through a multi-scale approach. In BP-Dock, the residues along a protein chain are perturbed mimicking the binding induced event, with a small Brownian kick, one at a time. The fluctuation response profile of the chain upon these perturbations is computed by Perturbation Response Scanning (PRS) to generate multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, this approach was applied to a large and diverse dataset of unbound structures as receptors. Furthermore, the protein-peptide docking of PICK1-PDZ proteins was investigated. This study elucidates the determinants of PICK1-PDZ binding that plays crucial roles in numerous neurodegenerative disorders. BP-Dock approach was also extended to the challenging problem of protein-glycan docking and applied to analyze the energetics of glycan recognition in Cyanovirin-N (CVN), a cyanobacterial lectin that inhibits HIV by binding to its highly glycosylated envelope protein gp120. This study provide the energetic contribution of the individual residues lining the binding pocket of CVN and explore the effect of structural flexibility in the hinge region of CVN on glycan binding, which are also verified experimentally. Overall, these successful applications of BP-Dock highlight the importance of modeling backbone flexibility in docking that can have important implications in defining the binding properties of protein-ligand interactions.
Finally, an induced fit docking approach called Adaptive BP-Dock is presented that allows both protein and ligand conformational sampling during the docking. Adaptive BP-Dock can provide a faster and efficient docking approach for the virtual screening of novel targets for rational drug design and aid our understanding of protein-ligand interactions.
A novel flexible docking approach called BP-Dock (Backbone Perturbation docking) was developed to overcome this challenge. BP-Dock integrates both backbone and side chain conformational changes of a protein through a multi-scale approach. In BP-Dock, the residues along a protein chain are perturbed mimicking the binding induced event, with a small Brownian kick, one at a time. The fluctuation response profile of the chain upon these perturbations is computed by Perturbation Response Scanning (PRS) to generate multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, this approach was applied to a large and diverse dataset of unbound structures as receptors. Furthermore, the protein-peptide docking of PICK1-PDZ proteins was investigated. This study elucidates the determinants of PICK1-PDZ binding that plays crucial roles in numerous neurodegenerative disorders. BP-Dock approach was also extended to the challenging problem of protein-glycan docking and applied to analyze the energetics of glycan recognition in Cyanovirin-N (CVN), a cyanobacterial lectin that inhibits HIV by binding to its highly glycosylated envelope protein gp120. This study provide the energetic contribution of the individual residues lining the binding pocket of CVN and explore the effect of structural flexibility in the hinge region of CVN on glycan binding, which are also verified experimentally. Overall, these successful applications of BP-Dock highlight the importance of modeling backbone flexibility in docking that can have important implications in defining the binding properties of protein-ligand interactions.
Finally, an induced fit docking approach called Adaptive BP-Dock is presented that allows both protein and ligand conformational sampling during the docking. Adaptive BP-Dock can provide a faster and efficient docking approach for the virtual screening of novel targets for rational drug design and aid our understanding of protein-ligand interactions.
ContributorsBolia, Ashini (Author) / Ozkan, Sefika Banu (Thesis advisor) / Ghirlanda, Giovanna (Thesis advisor) / Beckstein, Oliver (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2015
![156067-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-08/156067-Thumbnail%20Image.png?versionId=ANlRVam_cVwDbQRtzAhw8bYt9Ee6qBtw&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T123713Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=cbe2753ad99f4ec92f3f35351f9849e962663e32e5f3d92df3c45bf8a0905a4d&itok=JodiONFS)
Description
Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
ContributorsDiamos, Andy (Author) / Mason, Hugh S (Thesis advisor) / Mor, Tsafrir (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2017
![136289-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-05/136289-Thumbnail%20Image.png?versionId=7Z0EKvhCwsRYTItUWhLa0u0N4da7dPDr&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T144340Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=5b9c23db512c9efb55e71cbc5f68eb04f3d0527b9f975583299d83d9ab6341f8&itok=KSsPZv5j)
Description
The Intercellular Adhesion Molecule-1 (ICAM-1, known as CD54) is a cell surface type I transmembrane glycoprotein with a molecular weight of 85 to 110 kDa. The primary function of ICAM-1 is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 is used as a receptor for various pathogens such as rhinoviruses, coxsackievirus A21 and the malaria parasite Plasmodium falciparum. ICAM-1 contains five immunoglobulin (Ig) domains in its long N-terminal extracellular region, a hydrophobic transmembrane domain, and a small C-terminal cytoplasmic domain. The Ig domains 1-2 and Ig domains 3-4-5 have been crystallized separately and their structure solved, however the full ICAM-1 structure has not been solved. Because ICAM-1 appears to be important for the mediation of cell-to-cell communication in physiological and pathological conditions, gaining a structural understanding of the full-length membrane anchored ICAM-1 is desirable. In this context, we have transiently expressed a plant-optimized gene encoding human ICAM-1 in Nicotiana benthamiana plants using the MagnICON expression system. The plant produced ICAM-1 is forming aggregates according to previous data. Thus, the current extraction and purification protocols have been altered to include TCEP, a reducing agent. The protein was purified using TALON metal affinity resin and partially characterized using various biochemical techniques. Our results show that there is a reduction in aggregation formation with the use of TCEP.
ContributorsPatel, Heeral (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kannan, Latha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
![136311-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-05/136311-Thumbnail%20Image.png?versionId=xpXo36oOS05AVC_EMEJzb_knwZmnCklu&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T144744Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=b17d3e9d8f680d2d7b5709a79acbcac189201a3fdbda14def1f25bb1fa93aa71&itok=1N1jzFip)
Description
Lipid membranes are a key structure for many classes of viruses. Lipid membranes can be analyzed using the fluid mosaic model, which states that the phospholipid membrane has variable amounts of fluidity and key membrane proteins are presented in areas stabilized by cholesterol-enriched platforms called lipid rafts. This project aims to further the understanding of the importance of lipid rafts in measles virus (MV) infection and replication, which has not been extensively studied. In order to do this, an MV-susceptible cell line was treated with an anti-cholesterol compound before and after measles virus infection. I found that pre-infection treatments had a marginal effect upon measles cytopathic effect (syncytia formation) or replication. Twenty-four hours post-infection treatment had a deleterious effect on cell viability, but the replication/assembly of infectious units per cell decreased importantly and in dose-dependent manner. Furthermore, by measuring the susceptibility to neutralization of infectious particles obtained from MBCD treated cells, I determined the importance of lipid microdomain environment on the stability of infectious particles. Increased anti-cholesterol treatment enhanced the susceptibility of MV to neutralization. Future studies are proposed to assess the properties of cholesterol depleted viral infectious units.
ContributorsYkema, Matthew Ryan (Author) / Mor, Tsafrir (Thesis director) / Jacobs, Bertram (Committee member) / Julik, Emily (Committee member) / Barrett, The Honors College (Contributor) / Economics Program in CLAS (Contributor) / School of Life Sciences (Contributor)
Created2015-05
![136320-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-05/136320-Thumbnail%20Image.png?versionId=cLacYvbiYyfVlDuSRPqVWfRVyVjNEsLV&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T055136Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=aac00c1dca9a2b33f05a6700adea42f688ce4898dd9fd3990e689f807316ac26&itok=Ulk_VG9h)
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. 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
![136383-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-05/136383-Thumbnail%20Image.png?versionId=8mw7mU5tU5BCOrdQduIshTBHa4NeaKC.&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T054029Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=79ff0d217f5fe69770b9ed3e4e338882b2e14ff22b3b2dbb98b973c06182d93f&itok=8GIPQD6d)
Description
We, a team of students and faculty in the life sciences at Arizona State University (ASU), currently teach an Introduction to Biology course in a Level 5, or maximum-security unit with the support of the Arizona Department of Corrections and the Prison Education Program at ASU. This course aims to enhance current programs at the unit by offering inmates an opportunity to practice literacy and math skills, while also providing exposure to a new academic field (science, and specifically biology). Numerous studies, including a 2005 study from the Arizona Department of Corrections (ADC), have found that vocational programs, including prison education programs, reduce recidivism rates (ADC 2005, Esperian 2010, Jancic 1988, Steurer et al. 2001, Ubic 2002) and may provide additional benefits such as engagement with a world outside the justice system (Duguid 1992), the opportunity for inmates to revise personal patterns of rejecting education that they may regret, and the ability of inmate parents to deliberately set a good example for their children (Hall and Killacky 2008). Teaching in a maximum security prison unit poses special challenges, which include a prohibition on most outside materials (except paper), severe restrictions on student-teacher and student-student interactions, and the inability to perform any lab exercises except limited computer simulations. Lack of literature discussing theoretical and practical aspects of teaching science in such environment has prompted us to conduct an ongoing study to generate notes and recommendations from this class through the use of surveys, academic evaluation of students' work and ongoing feedback from both teachers and students to inform teaching practices in future science classes in high-security prison units.
ContributorsLarson, Anika Jade (Author) / Mor, Tsafrir (Thesis director) / Brownell, Sara (Committee member) / Lockard, Joe (Committee member) / Barrett, The Honors College (Contributor) / School of Politics and Global Studies (Contributor) / School of Life Sciences (Contributor)
Created2015-05
![133792-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-05/133792-Thumbnail%20Image.png?versionId=t7WBD1Ev9gYZlSOsq1HNk.t0KPCm0.WI&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T112725Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=c81e5f2bfbbdb4a0019585506cee9ddb1690034a3a62b6cb3a7b4a510292209e&itok=dYsFG1pG)
Description
A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium tumefaciens infiltration. Characterization and expression studies were then conducted to confirm correct assembly of the antibody. Once the antibody was purified, an ELISA was conducted to validate that the antibody was able to bind to the flavivirus fusion loop.
ContributorsPardhe, Mary (Author) / Mason, Hugh (Thesis director) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / School of Life Sciences (Contributor) / Department of Information Systems (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
![133844-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-05/133844-Thumbnail%20Image.png?versionId=QLOa33lU8TEj7qY2oXgLuyqo_2nNMtzH&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240426/us-west-2/s3/aws4_request&X-Amz-Date=20240426T054029Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=1bd3ba813141454bc5a62402a4c3d24817bc072490944c14280a4fc885c44312&itok=DXrMk84y)
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 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