Browsing by Subject "Protein"
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Item Cloning, expression, and purification of Burkholderia protein targets for diagnostic and vaccine development(2012-05) McCaul, Kate Christina; Brown, Katherine A.; Kitto, George B.Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. These diseases are endemic mainly in southeastern Asia and northern Australia, but they also pose a bioterrorism threat in the developed world. These diseases have high mortality, partially due to the lack of vaccines and rapid, accurate diagnostic assays. The work discussed here represents a part of a larger project to develop a dependable diagnostic assay for use in both developing endemic areas and the developed world, as well as a subunit vaccine to protect against disease. In this study, several proteins from B. pseudomallei, B. mallei, and the closely related but less virulent B. thailandensis have been cloned, expressed and purified in order to develop highly sensitive and specific diagnostic reagents for the detection of B. pseudomallei and B. mallei in infected patient samples. Protein targets expressed in this study were also used in subunit vaccine development for melioidosis and glanders.Item Cytoplasmic foci at the crossroads of artifactual science and biological function(2016-05) Zhao, Alice; Marcotte, Edward M.; Ellington, Andrew D; Zhang, Yan J; Appling, Dean R; Iyer, Vishwanath RDeciphering protein interaction and compartmentalization is crucial to understanding the molecular mechanisms that drive biological processes. Using various high throughput approaches, we have managed to score subcellular dynamic protein re-organization into supramolecular structures and map physical association networks to discover protein complexes on a proteome-wide level. However, the case by case studies of some of these novel structures and interactions reveal difficulties in interpreting their biological basis. This study offers insights into limits inherent in the molecular techniques used to investigate subcellular structures and protein interactions, describing a set of cautionary tales and critical analysis for deciphering cases of confounding data from orthogonal approaches. This study also offers a new experimental technique for high-throughput imaging assays with mammalian cell lines.Item Design and synthesis of conformationally constrained ligands for Grb2 SH2 binding and thermodynamic evaluation and the development of a diversity oriented synthesis of 2-arylpiperidines(2016-12) Goodnough, Alex Michael; Martin, Stephen F.The ways in which torsional strain in the bound form of a ligand affects the energetics of protein binding are poorly understood. In order to study this feature of protein-ligand interactions, a conformationally constrained ligand for Grb2 SH2 containing a 1,1,2 trisubstituted cyclopropane was designed, and the synthesis of this ligand attempted. Additionally, a novel iminium ion formation/cyclization cascade was applied to the synthesis of a library of 2-arylpiperidines with varying aryl group substitution, and nitrogen atom functionalization. This strategy should allow further access to chemical space already identified as containing potential therapeutics and tool compounds for biological interrogation.Item Development of dry powder inhalation for biological drug modalities(2020-05) Zhang, Yajie; Williams III, Robert O., III, 1956-; Watts, Alan B.; Smyth, Hugh D.C.; Cui, Zhengrong; Ghosh, DebadyutiDry powder inhalers (DPI) have been developed to topically deliver therapeutics to the lung. Generally, a particle size range of 1-5 µm is desirable for the lung deposition of drug aerosols. To date, several particle engineering technologies have been developed and successfully applied to micronize powders of small molecule drugs. However, particle size reduction is challenging for biological materials due to their ductile texture and susceptibility to stresses encountered during processing. In this dissertation, three biological drug modalities (i.e. peptide, protein, and bacteriophage), are micronized using different particle engineering technologies. Chapter One provides a review of formulations and delivery systems used in peptide therapeutic products. Chapter Two describes several stages in the development of an excipient-free peptide inhalation product, including pre-formulation, formulation and processing, stability testing, and animal studies. It was demonstrated that jet milling is effective in manufacturing a stable, excipient-free peptide inhalation powder for the treatment of pulmonary fibrosis. In Chapter Three, inhalable dornase alfa powder is formulated with various excipients and the formulations are processed using thin film freeze-drying (TFFD) technology. The excipient screening was performed by employing design of experiment (DoE). Formulations that were both inhalable and preserve the enzymic activity of the protein were found. In Chapter Four, a review of inactive ingredients used in solid bacteriophage formulations is presented. Finally, Chapter Five explores the feasibility of using TFFD to manufacture inhalable bacteriophage powders. It was proved that TFFD is a suitable technology to produce powders that have desirable properties, including preserved bioactivity of bacteriophage, inhalable size range, and other physical characteristics.Item The effects of carbohydrate-protein supplementation on glycogen utilization and fatigue during a simulated soccer match(2010-12) Dessard, Benjamin; Ivy, John, 1945-; Tanaka, HirofumiThe purpose of this study was to examine if the addition of protein to a carbohydrate supplement (CHO+PRO), provided during a simulated soccer match, would reduce fatigue and muscle glycogen utilization in comparison to an isocaloric carbohydrate only supplement (CHO). Two female and eight male (n = 10) trained soccer players performed a modified version of the Loughborough Intermittent Shuttle Test (LIST) on two separate occasions, followed by a run to exhaustion (RTE). Supplements were provided 10 minutes before the simulated match and at the beginning of half-time, but not during exercise in order to create real-match conditions. Supplements were composed of 2.8% protein + 7% carbohydrate (CHO+PRO) or 9.8% carbohydrate (CHO). Muscle biopsies were performed before and at the end of the LIST, after which iv participants ran to exhaustion. No differences were found between treatments for RTE (489 ± 121 sec for CHO and 589 ± 186 sec for CHO+PRO) or glycogen utilization (37.9 ± 7.6 µmol•g wet wt-1 during the CHO and 29.1 ± 6.0 µmol•g wet wt-1 during the CHO+PRO). No differences were found for the other measurements such as sprint times, heart rate, RPE, blood glucose, lactate, and insulin. Blood Creatine kinase (CK), and overall muscle soreness were measured 24 hours after each trial in order to evaluate muscle damage but no differences between treatments were found. In accordance with these findings, the phosphorylation state of the protein FOXO3a was not altered differently by the treatments. These results suggest that the addition of protein to a traditional carbohydrate-only supplement provided immediately prior to and at the half of a simulated soccer match does not further improve the benefits of a CHO supplement.Item Exploring N- and C-terminal Fragment Ion Biases in UV-Photodissociation Mass Spectrometry of Intact Proteins(2024) Lam, Raymond; Brodbelt, Jennifer S.193 nm UV-photodissociation (UVPD) is a powerful ion activation method in tandem mass spectrometry (MS/MS) for analyzing complex biomolecules and proteins. Sample ions are isolated by their mass-to-charge m/z ratio and exposed to pulsed UV light, causing absorption of UV photons and cleavage along the amide backbone to generate informative fragment ions. The high energy deposition of UVPD and preservation of higher-order structure and modifications makes it particularly appealing for analysis of large, modified, heterogeneous, or multimeric protein states that prove challenging for conventional tandem mass spectrometry methods. It is expected that fragment ions containing the N- and C-terminal ends of each protein ion should be produced and detected equally upon amide backbone cleavage. Data among recent 193 nm UVPD-MS analyses and other MS/MS methods (such as collision-induced dissociation) show bias in the production of N- and C-terminal ions in different m/z mass-to-charge regions of mass spectra. Owing to the development of UVPD for top-down protein analysis, this method was examined in more detail for the N-term/C-term bias and was the focus of the present study. Among a set of six proteins prepared in denaturing solution conditions to generate standard and “supercharged” charge states, fragment ion identifications from proteins with greater numbers of basic residues were biased towards N-terminal ions in lower m/z regions, while proteins with acidic residues biased C-terminus-containing fragment ions in the same region. The backbone sites of generated fragment ions showed precursor charge-state dependence on the degree of ion current bias but remained intrinsically biased towards N/C-terminal fragment ions, while explorations of the charge of fragment ions proved insufficient to provide rationale for the migration of fragment ions into different m/z regions. This promotes further study of biases in top down MS/MS analysis of proteins, particularly as applied to MS techniques attempting to resolve information from spectrally noisy yet information-rich UVPD fragment ions, like proton-transfer charge reduction and internal fragment ion assignment.Item Functional analysis of DdINCENP, a chromosomal passenger protein, in Dictyostelium(2006-08) Chen, Qian, 1975-; De Lozanne, ArturoDictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. The localization of DdINCENP at the cleavage furrow is modulated by myosin II. Loss of myosin II restricted the localization of DdINCENP to a narrow zone at the cleavage furrow. Kif12, a homolog of mitotic kinesin like protein (MKLP), was essential for relocalization of DdINCENP from the central spindle to the cleavage furrow. Furthermore, Kif12 was also localized at the cortex of the cleavage furrow and its localization during cytokinesis closely resembled that of DdINCENP, suggesting a possible interaction between them. The correct localization of DdINCENP during cytokinesis also required its N-terminal sequence. DdINCENP1-500 was found at the cleavage furrow and interacted with the actin cytoskeleton. Domain analysis of DdINCENP also revealed that its DdINCENP1-500 was sufficient to rescue the weak spindle defect of DdINCENP null cells.Item Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli(2014-05) Rodríguez Mendoz, Álvaro Eugenio; Barrick, Jeffrey E.; Harshey, RasikaSynthetic biologists and metabolic engineers seek to design and create organisms with novel functions. A major difficulty with many designed genetic devices is that they lack evolutionary robustness. In this study, our aim was to identify mutations that could enhance the evolutionary stability of green fluorescent protein (GFP) expression from a plasmid in Escherichia coli. To achieve this goal, we created a mutagenized strain library and performed an evolution experiment. To enrich potential mutants with improved GFP stability, we periodically sorted for cells that remained highly fluorescent as this population was propagated for several hundred generations and less-robust strains accumulated inactivating mutations. Further testing of clones isolated from the final evolved population showed that GFP expression was more stable in these strains and suggested mutations in the chromosome were responsible. Re-sequencing the genomes of four of these strains found that, among other genetic differences from the ancestor, all had a mutation in either PolA or PolB. These two types of DNA polymerase mutations may enhance GFP stability by causing a lower point mutation rate in the E. coli host.Item Isolation and characterization of Pisum sativum apyrases, PsNTP9 and PsNTP9-DM, cloned and expressed in Escherichia coli(2019-02-06) Wallen, Michael Andrew, Jr; Roux, Stanley J.Adenosine triphosphate (ATP) is widely known as a fuel source for many biochemical processes, and to a lesser degree also as a signaling molecule in plants and animals. When plants are subjected to biotic or abiotic stress or undergoing exocytosis, they release ATP into the extracellular matrix (ECM). The release of ATP sets off a signal transduction pathway, first rapidly increasing the concentrations of cytosolic calcium, reactive oxygen species, and nitric oxide. How these changes specifically influence physiology is the object of much research in both plants and animals. Some of the changes that are affected influence growth and development, stomatal function, and gravitropism. Apyrases and other phosphatases control the concentration of the released nucleotides by breaking phosphate bonds from nucleoside triphosphates and diphosphates. Research aimed at the discovery of receptors, signaling pathway components, and processes has been successful to some extent. There are now known purinergic receptors in both plants and animal cells. We have cloned a truncated version of Pisum sativum (ps) NTP9. We used a pET-22B vector to add a histidine tag and transformed the vector into the BL21 Escherichia coli with a T7 promoter to enable IPTG induction of the LAC operon and expression of the enzyme. The pET-22B vector was incubated in separate samples with BL21 cells. Cells were propagated, and the expression of recombinant proteins PsNTP9, and separately, a double mutant PsNTP9-DM with a second calmodulin-binding domain, were induced ectopically. Cells were broken open by shaking them and mixing them with lysis buffer. Centrifugation was performed to separate the supernatant containing the released apyrases from the particulate wall fraction. The enzymes were purified by affinity chromatography, then their purity was evaluated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Western blots were performed to verify presence of the apyrases using a commercial anti-histidine antibody to detect PsNTP9 and PsNTP9-DM. Once suitable amounts of our proteins of interest were harvested, we performed Bradford assays to determine the protein concentration of the samples and carried out an apyrase activity assay to determine the specific activity of the purified enzymes and compare it to that of other known phosphatases.Item The manufacture and characterization of protein nanoclusters(2013-05) Dinin, Aileen Kathryn; Johnston, Keith P., 1955-; Maynard, Jennifer Anne, 1974-The ability to formulate monoclonal antibodies at high concentration in a low-viscosity form is of broad interest in drug delivery, as monoclonal antibody-based drugs are now prescribed for cancer, autoimmune disorders, and many other diseases. Herein, we create highly concentrated antibody dispersions (up to 260 mg/mL) via three different methods, utilizing proline as an interacting depletant or trehalose as a non-interacting depletant. These dispersions are able to achieve viscosities an order of magnitude lower than similarly concentrated antibody solutions over a range of formulation pHs. When diluted, these antibody dispersions return to monomer. The proline acts to minimize protein zeta potential, thus reducing the electrostatic repulsion on the protein, even when formulated 3 pH units away from the antibody pI. In addition, it acts as a depletant, forcing the monomers into cluster via osmotic effectsItem Modeling the structure, dynamics, and interactions of biological molecules(2013-05) Xia, Zhen, active 2013; Ren, PengyuBiological molecules are essential parts of organisms and participate in a variety of biological processes within cells. Understanding the relationship between sequence, structure, and function of biological molecules are of fundamental importance in life science and the health care industry. In this dissertation, a multi-scale approach was utilized to develop coarse-grained molecular models for protein and RNA simulations. By simplifying the atomistic representation of a biomolecular system, the coarse-grained approach enables the molecular dynamics simulations to reveal the biological processes, which occur on the time and length scales that are inaccessible to the all-atom models. For RNA, an "intermediate" coarse-grained model was proposed to provide both accuracy and efficiency for RNA 3D structure modeling and prediction. The overall potential parameters were derived based on structural statistics sampled from experimental structures. For protein, a general, transferable coarse-grain framework based on the Gay-Berne potential and electrostatic point multipole expansion was developed for polypeptide simulations. Next, an advanced atomistic model was developed to model electrostatic interaction with high resolution and incorporates electronic polarization effect that is ignored in conventional atomistic models. The last part of my thesis work involves applying all-atom molecular simulations to address important questions and problems in biophysics and structural biology. For example, the interaction between protein and miRNA, the recognition mechanism of antigen and antibody, and the structure dynamics of protein in mixed denaturants.Item Protein Denaturation in Complex Solvents: Using FTIR Spectroscopy to Understand Cryopreservation(2020) Rajesh, Kavya; Baiz, Carlosprotectants are commonly used to stop ice formation in biological systems, allowing for long term preservation and storage. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however; it can also be toxic to cells which limits the use of DMSO in organs. Modulating the toxicity of DMSO could aid in long term organ storage and cryopreservation, creating organ banks and dramatically reducing the size of the organ donation waitlist. Cryoprotectant toxicity neutralizers, including formamide, have been shown to aid in neutralizing the toxicity of cryoprotectants such as DMSO. However, organs are large and multifaceted which presents a host of experimental challenges. Simple proteins like lysozyme can be used to model complex organ systems. Here we use amide I infrared spectroscopy to measure thermal denaturation curves of lysozyme in ternary mixtures of DMSO, water, and formamide or dimethylformamide. We show that formamide increases the stability of lysozyme in DMSO/water mixtures, suggesting that it protects against DMSO denaturation. We also show the effects of other amides like N-methylacetamide and N-methylformamide on lysozyme. The structures of these co-solvents are shown in Figure 1. Based on their differing structures and hydrogen bonding capabilities, these amides modulate the toxicity of DMSO in different ways. This information can be used to create mixtures of co-solvents to mitigate DMSO toxicity and eventually cryopreserve biological systems.Item Relation of protein-protein interactions to rheological properties and stability of highly concentrated of monoclonal antibodies(2018-08-16) Dear, Barton Joseph; Johnston, Keith P., 1955-; Truskett, Thomas Michael, 1973-; Lynd, Nathaniel; Stachowiak, JeanneSolutions of monoclonal antibodies (mAbs) at high concentration are strongly desirable for subcutaneous delivery for the treatment of many autoimmune diseases and cancer. However, at the high concentrations necessary for practical application, mAbs tend to have strongly attractive protein-protein interactions (PPI) that cause the solutions to be highly viscous and prone to aggregation. Herein, the PPI of various mAbs and other proteins at concentrations up to 250 mg/ml are tuned by the addition of the small molecule co-solutes that can screen both electrostatic and hydrophobic PPI to produce low viscosities. The effects of the co-solutes on PPI were directly measured using small angle x-ray scattering, dynamic light scattering, and shear-dependent rheology as well as indirectly through the viscosity and storage stability. MAb solutions with attractive net PPI will be shown to be generally more viscous and aggregation prone than solutions with less attractive net PPI; however, these correlations remain challenging to understand across mAbs. Alternatively, the formation of reversible oligomers, or clusters in solution, as determined by fitting SAXS structure factors with molecular dynamic simulations, or the polydispersity of DLS data, will be demonstrated to be a key factor in determining high concentration viscosity. The storage stability will be shown to be influenced by both PPI and conformational stability; therefore, co-solutes that improve both will cause yield the highest stabilities.Item Simultaneous, single-carrier delivery of antigens and immune-modulatory molecules to dendritic cells(2013-05) Dawson, Eileen Regina; Roy, Krishnendu; Peppas, Nicholas A., 1948-Immunotherapy as a means for cancer treatment has been investigated for over a century. While studies have been completed using different immunological strategies, development of a clinical therapeutic cancer vaccine has proven elusive. Recently, success has been seen with prophylactic vaccines for cancers with known viral origins (Gardasil® and Cervarix for Human Papiloma Virus). However, such strategies do not address the challenge in generating effective immune response against other tumor antigens, most of which are weakly immunogenic self-antigens. Tolerance to these self-antigens could ultimately limit the patient’s ability to mount an effective anti-tumor immune response. The US Food and Drug Administration recently approved the first DC cell-based cancer vaccine, Provenge®, for use in prostate cancer. This vaccine requires cell isolations from the patient as well as in vitro DC modifications, which ultimately leads to high cost as well as multiple procedures. However, results indicate that, on average, patients live only four months longer than those receiving a placebo. While this work remains important, and offers proof that priming DCs can improve the lifespan of a patient, it ultimately does not offer a long-term cure. Direct and highly efficient in vivo delivery of antigens to DCs could overcome the challenges associated with ex vivo DC manipulation and may offer a more scalable method for generating anti-tumor immunity. This research focuses on the development of novel formulations that allow simultaneous delivery of protein/peptide-based tumor antigens and immune-modulatory nucleic acids (siRNA and immune stimulatory CpG) to the same dendritic cells (DCs) in-vivo. Such formulations allow a synthetic immune-priming center to be created at the site of immunization and simultaneously deliver the tumor antigen to DCs and modulate their immune response through IL-10 silencing. Our hypothesis is that using such a DC-targeted dual delivery system we will be able to illicit strong T helper 1 (TH1) and Cytotxic T Lymphocyte (CTL) response in vivo against a wide array of tumor antigens. This can become a platform technology where the biomolecules (antigen and immunomodulatory agents) can be easily varied based on particular cancers.Item Structure-function analysis of a Group II Intron reverse transcriptase(2022-05-09) Alvarado Torres, Jose Mario; Lambowitz, Alan; Russell, RickMobile group II introns are ribozymes and mobile genetic elements found in all branches of life. They consist of a highly structured intron RNA that encodes a reverse transcriptase (RT). This RT is utilized during retrohoming to synthesize a full-length DNA copy (cDNA) of the intron RNA which is then integrated into new genome locations. One theory suggests that RTs evolved from an RNA Dependent RNA Polymerase (RDRP) that originated in the RNA World. These ancestral RDRPs served to propagate genetic material, and during the transition to DNA-based life, RDRPs evolved into RTs. Recent structural data for Geobacillus stearothermophilus Group II Intron RT (GsI-IIC RT) suggests a close evolutionary relationship between RDRPs and group II intron RTs, including a strikingly similar RT active site and regions that function in binding the template and primer. To further probe this evolutionary relationship rational mutations were introduced into GsI-IIC RT that affected rNTP incorporation ability. Group II introns have also potentially evolved characteristics like higher fidelity and processivity compared to other RTs due to stringent accuracy requirements when reverse transcribing the catalytically active and highly structured intron RNA. Host-encoded group II intron-like RTs that have acquired other functions often have substitutions for the conserved A residue in the YADD motif at the RT active site. This suggests that the active site can be modified by evolutionary pressures to suit diverse activities. This tolerance was exploited to mutate the highly conserved YADD motif to study structure-function relationships between conserved features of previously uncharacterized classes of RTs. Current laboratory data suggests that the YXDD motif can be modified to modulate different activities, although other crucial complementary or compensatory mutations are likely necessary. Through this work I obtained data that suggests group II intron RTs evolved from ancestral RdRPs, that their constricted active site might serve to increase fidelity, and that their conserved active site motif can be modified to modulate biochemical activities.Item The effects of carbohydrate and amino acids on muscle protein synthesis after acute resistance exercise and muscle adaptation following chronic resistance training(2016-05) Wang, Wanyi; Farrar, Roger P.; Ivy, John, 1945-; Brothers, Robert M; Wilcox, Richard E; Jolly, Christopher A; Bray, Molly SResistance exercise (RE) is purported to induce muscle protein accretion primarily by stimulating muscle protein synthesis (MPS), with its effect potentiated by providing a protein or amino acid (AA) supplement post exercise. Glutamine, a conditionally essential AA, is increasingly recommended to improve exercise performance, but it is poorly soluble and unstable in sports drinks. This limitation can be overcome by combining L-glutamine with L-alanine to form a dipeptide (AlaGln). The first study demonstrated that AlaGln supplementation post resistance exercise significantly reduced the phosphorylation of AMPK and NF-kB p65 without activating intracellular signals for MPS. This study also showed that whey protein (WP) activated the mTOR signaling pathways without affecting signaling proteins that controls muscle protein breakdown (MPB). The results provide indirect evidence that AlaGln blocks MPB via suppressing the activation of AMPK-FOXO3A and NF-kB p65, while WP promotes MPS. Recent research suggests that adding carbohydrate (CHO) to a protein supplement post exercise can produce greater protein accretion and subsequently increase the magnitude of resistance training adaptation relative to protein supplementation alone. Early studies from our laboratory found that co-ingestion of CHO and protein, as compared with CHO or protein intake individually, had a greater effect on the activation of anabolic signaling proteins. However, the effect of CHO plus protein on MPS and muscle adaptation is controversial. The second study demonstrated that adding CHO to a protein supplement (CP) accelerated MPS via activating the mTOR-signaling pathway in comparison with placebo (PLA) and WP during early exercise recovery, but CP did not affect signaling proteins that regulate MPB. The third study found that CP enhanced muscle strength relative to PLA and WP. The greater strength development in CP appeared due to increased myofibrillar protein content. Increased muscle hypertrophy occurred without an increase in myonuclei suggesting satellite cell activation was not required for muscle fiber development. Taken together, the results of this series of studies suggest that 1) AlaGln inhibits MPB following acute RE; 2) Adding CHO to a protein supplement accelerates muscle recovery by stimulating MPS, and increases the magnitude of muscle strength by accumulating more myofibrillar protein in comparison with PLA and WP.