Browsing by Subject "Protein"

Now showing 1 - 17 of 17
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    2018 Undergraduate Research Symposium Presentation on Method for Detection for Protein Aggregation Propensity
    (2018) Fransen, Katharina A; Ritter, Seth C; Hackel, Benjamin
    Engineered proteins are valuable tools for clinical therapeutics and diagnostics as well as for other biotechnology applications. The physicochemical robustness of a protein is an important factor in its utility. For example, protein solubility is advantageous for production, conjugation, formulation, and use; yet numerous engineered proteins exhibit suboptimal solubility due to the formation of protein aggregates. For the technological aim of identifying superior reagents and the scientific goal of elucidating protein sequence – function relationships, we are developing a method for high-throughput analysis of protein aggregation. The method uses yeast surface display in which the protein of interest (POI) is coupled to the surface of a S. cerevisiae yeast cell via an extended ‘PAS40’ polypeptide linker to the native yeast mating protein Aga2p, which binds to the yeast-bound Aga1 membrane protein. Meanwhile, the DNA encoding the POI is retained inside the yeast thereby creating a phenotype-genotype linkage that enables independent evaluation of millions of POI variants with simple DNA sequencing to reveal the identity of functional variants after aggregation analysis. Aggregation analysis utilizes Foerster resonance energy transfer (FRET) in which an excited donor fluorophore transfers energy to a nearby acceptor fluorophore. The acceptor and donor fluorophores are attached near the N-terminus of the POIs, allowing aggregating POIs to bring the fluorophores within the necessary radius for energy transfer and subsequent acceptor fluorescence. Measurement of the fluorescence of the acceptor protein allows for the aggregation analysis of the POI, which can be performed in high-throughput (100 million per hour) via flow cytometry.
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    Characterizing the flow behavior of protein and excipient solutions using Dripping-onto-Substrate extensional rheometry
    (2023-02) Lauser, Kathleen
    Injectable protein medications are lifesaving therapies for patients with cancer, COVID-19, and autoimmune diseases. However developing stable, concentrated protein therapies canbe challenging due to regulations requiring small volumes and viscosities. Accordingly,many therapies are administered intravenously at hospitals, requiring long, expensive stays.Developing ultra-concentrated (>150 mg/mL) medications that can be self-administered subcutaneously can reduce costs and improve flexibility for patients. However ultra-concentrated formulations often suffer from high viscosities and poor stability at rest and under flow. Further, protein medications can denature and lose efficacy when injected due to strong extensional “stretching” forces. These extensional flows can be more detrimental to protein structure and function than shear flows, although thorough studies of protein extensional rheology are limited due to volume constraints and instrumentation challenges. Further, pharmaceutical excipients – molecules like polymers or surfactants which are added to reduce shear viscosity and stabilize formulations – can produce complex flow effects in extension. The first goal of this thesis is to build and validate a novel instrument to measure extensional rheology and associated material properties of protein and protein-excipient solutions for the first time. To do so, a modified dripping-onto-substrate (DoS) extensional rheology device was designed and created. DoS is an extensional rheological technique that creates a semi-stable liquid bridge from a single drop. In time, the liquid bridge self-thins due to inertial, surface tension, viscous and elastic forces. The evolution of the liquid bridge radius is captured with high-speed imaging, which can then be fit to extract rheological parameters. The modified DoS instrument enables measurements of low-viscosity solutions in pure extensional flows in 10 μL or less, overcoming traditional limitations associated with measuring protein solutions in extension. This technique was validated using several test fluids with well-known literature values prior to measuring protein solutions. The second goal was to understand the flow behavior and potential synergistic or antagonistic effects between proteins and pharmaceutical excipients in extensional flows. Model protein ovalbumin (OVA) solutions with added FDA-approved excipients poloxamer 188 (P188), polysorbate 20 (PS20), or polysorbate 80 (PS80) were examined using the DoS technique. OVA is similar in size to insulin, which has previously demonstrated injection-dependant flow behavior. However promisingly, OVA-only solutions up to 300 mg/mL protein exhibited rapid thinning and breakup behavior characteristic of low viscosity fluids. Conversely, excipients typically added to prevent protein aggregation at restor in shear flow appeared to cause detrimental behavior in injection-like flows. P188, a poloxamer that is primarily composed of unimers in solution, demonstrated rapid thinning at low concentrations but transitioned to weakly elastic behavior at higher concentrations. P188 addition was required to observe elasticity in combined P188/OVA conditions since OVA alone did not demonstrate elasticity at any studied concentration. Compared to P188, PS20, and PS80 are smaller molecules and form micelles at the studied conditions. Although PS20 and PS80 are structurally similar, differences in surface activity result in observed flow differences at low concentrations for PS and PS/OVA solutions. However, at higher concentrations, PS20 and PS80 behavior becomes statistically identical due to crowded solution effects. The final goal was to examine the effect of substrate spreading behaviors on capillary-driven thinning, which is not well-explored, particularly for low-viscosity solutions. While capillary-driven thinning progresses in DoS experiments, fluid spreads on the substrate; these differences in spreading or other instrument parameters can lead to variations in the liquid bridge breakup times and rheological parameters. These discrepancies in behavior can be reconciled by correlating to the dimensionless Weber number as well as other fluid and instrument parameters such as aspect ratio or drop volume. Further, computing spreading distances and Weber numbers of spreading can elucidate the importance of Marangoni stresses in capillary thinning experiments for surface-active macromolecules. The results of this thesis demonstrate the first capillary thinning rheological measurements of protein excipient solutions and create a methodology for measuring future protein/excipient combinations. Identifying differences in flow behavior between formulations is important for pharmaceutical development to create more stable therapies in extensional flows. Additionally, an understanding of capillary thinning and spreading dependence can explain variation in DoS experiments and lead to more accurate comparisons of experiment results across samples and concentrations. While this thesis focused on protein-excipient solutions, many of the leanings on methodology are generally relevant to low-viscosity fluids, which can be useful for the broader rheological community.
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    Covalent Reactions Between Flavors and the Model Protein β-Lactoglobulin
    (2020-07) Anantharamkrishnan, Vaidhyanathan
    Demand for high protein plant and dairy-based diets has been increasing but delivering them has become problematic for the food industry. The flavor issues are due to multifaceted interactions that occur between food proteins and flavoring components. Over 40 years of research has been done on studying temporary interactions between flavor and proteins, but very little work has been done on more permanent interactions – covalent bonding. Covalent bonding takes place between the side chains and terminal amino acids of food proteins and reactive aroma compounds that will change the flavor profile of the product in a permanent manner. β- lactoglobulin (BLG) was chosen as a model protein for this study as it is well characterized in both amino acid sequence and structure, its molecular weight is suitable for intact protein mass spectrometry and it is a major protein used in food industry. This thesis study developed a methodology using UPLC-ESI/qTOF-MS for monitoring the nature and extent of the covalent reactions based upon the change in molecular weight (Protein + flavor) that occurs after reaction. The cross linking of protein with flavor compounds was evaluated using gel electrophoresis. A proteomics approach using LC and tandem MS after enzymatic digestion was taken to identify the sites of post-translational modification between the flavor compounds and the BLG protein. The UPLC-ESI/qTOF-MS methodology in tandem with proteomics and gel electrophoresis yield a detailed view of flavor/BLG interactions that offered insights on minimizing these undesirable reactions in the future. A flavoring typically is created from a mixture of volatile chemicals that generally comprise a variety of functional groups. Some of these flavor components when added to a protein matrix form covalent adducts resulting in a change in flavor character or a loss of its potency. The end result of these reactions create an imbalanced flavor, one that is not acceptable to a consumer. This research study analyzed 47 different flavor compounds from 13 different functional groups for their covalent adduct formation with BLG. Aldehydes, sulfur-containing molecules (especially thiols), and functional group-containing furans were found to be the most reactive of the flavor components studied. Thiol-containing compounds reduced disulfide linkages in BLG to result in disulfide interchange and formation of new disulfide linkages with the free cysteine group. Ketones were generally stable, but α-diketones (e.g., diacetyl) were reactive. Some bases (e.g., pyrazines and pyridines) were non-reactive, while the nucleophilic allylamine was reactive. Hydrocarbons, alcohols, acids, esters, lactones, and pyrans did not give observable levels of adduct formation within the time period studied. Due to the varied environmental conditions present in various food systems, the nature and extent of covalent interactions would likely change. This study investigated the influence of pH, temperature and water activity on the covalent adduct formation between BLG and selected flavor molecules. Covalent adduct formation was slower in acidic pHs. The rate and extent of the reaction increased with increasing pH. The rate of formation of adducts increased with temperature. Higher temperatures (45°C) caused the formation of products that were not observed at lower temperatures (4°C and 25°C). An increase in water activity lead to an increase in formation of adducts for allyl isothiocyanate. There were no observable differences for the effect of water activity on the reaction rate for benzaldehyde, citral and dimethyl disulfide. Results will help in understanding the conditions at which flavor compounds will covalently bond with a protein and ways to potentially avoid it. Thereby, helping the food industry to develop flavor protein matrices that have a longer shelf life.
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    A DC-STAMP domain within C. elegans sperm protein SPE-42 is required for fertilization
    (2013-08) Pung, Janel
    New life requires a sperm and an egg to progress through stages of recognition, binding and fusion once they meet at the site of fertilization. Relatively little is known regarding the common molecular mechanisms of fertilization that unite all metazoans as well as the divergences that are necessary for species specificity. The nematode C. elegans has been instrumental in the discovery of several sperm and egg genes that are required for fertilization. Spermatogenesis defective (spe) gene spe-42 functions at the time gametes meet. spe-42 mutant sperm look and behave like wild type sperm, but fail to fertilize oocytes. The spe-42 family is present in all organisms that use sperm and eggs and thus may be evolutionarily as old as the sperm and egg system itself. SPE-42 is predicted to be a six-pass sperm plasma membrane protein and contains three essential domains, a large extracellular domain, a RING finger domain, and a dendritic cell-specific transmembrane protein (DC-STAMP) domain. The original DC-STAMP protein is required for cell-cell fusion events unrelated to fertilization such as fusion of preosteoclasts into osteoclasts. The presence of DC-STAMP domains in these otherwise unrelated protein families suggests that this domain is involved in the mediation of membrane fusion. Amino acids within the SPE-42 DC-STAMP domain that showed conservation among the many DC-STAMP domains analyzed were mutated to explore the effect on protein activity. One amino acid was shown to be absolutely essential for function, 2 amino acids nearly erased function and 4 showed mild effects on function. One triple amino acid substitution had no effect on protein function. We also showed that the homologous C. briggsae SPE-42 DC-STAMP domain is functional within C. elegans SPE-42 DC-STAMP domain. These results support our hypothesis that the DC-STAMP domain is required for SPE- 42 function and suggest it is also critical for membrane fusion events mediated by the canonical DC-STAMP protein.
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    Developing methods to understand and engineer protease cleavage specificity
    (2016-09) Lane, Michael
    Proteases are ubiquitous enzymes that comprise nearly 2% of all human genes. These robust enzymes are attractive potential therapeutics due to their catalytic turnover and capability for exquisite specificity. While most existing drugs require a stoichiometric ratio to function, therapeutic proteases could clear their targets much more efficiently. Unfortunately, existing technologies are inadequate for understanding and engineering therapeutic proteolytic specificities. My thesis work has focused on building the groundwork to enable these technologies to thrive. For the goal of engineering a new protease, it is currently necessary to identify prototype proteases for engineering efforts that have specificities similar to the desired target substrate. Current technologies are unable to characterize proteases adequately for this goal. Accordingly, I invested in developing a method for the accurate characterization of protease cleavage specificity. Our unique combination of mRNA display technology, Next-Generation Sequencing, and mass spectrometry enables the sampling of all possible permutations of octamer substrates and the identification of millions of cleavage sites. The throughput of our approach is orders of magnitude greater than the current state-of-the-art methods. The resulting high-resolution specificity maps can be applied to identify promising protease prototypes, predict human cross-reactivity, or lead to a better understanding of this critical component of natural physiology. In the work presented here, I applied my new specificity-screening method to assess the specificities of the proteases factor Xa, ADAM17, and streptopain. The resulting cleavage preference maps confirmed known specificities, and revealed new insight into the broad preferences of both narrow- and broad-specificity proteases. In particular, disfavored amino acids were illuminated better than ever before. The next focus of my work was to engineer multiple-subsite novel protease specificity. I chose streptopain as the prototype for my efforts to neutralize the superantigen exotoxin SpeA. I identified a target loop of SpeA wherein cleavage would result in inactive fragments. Further, I confirmed that streptopain can be successfully presented as an mRNA displayed fusion. In summary, my thesis work established crucial methodologies for applying mRNA display technology to enable the understanding and ultimately engineering the specificity of therapeutic proteases.
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    Dietary protein and weight in midlife adults
    (2013-04) Aldrich, Noel David
    Current dietary recommendations include 0.8 g/kg/day of protein to meet metabolic requirements of nitrogen and amino acids; however, a growing body of data has identified positive changes in weight, body composition, blood pressure, and metabolic markers with increased intake of protein. Related to increased protein intake, research has been directed toward various sources of protein and composition responsible for specific metabolic responses. Therefore, more research is needed to evaluate the effective use of protein to improve body weight and composition, and perceptions consumers have regarding the role of dietary protein in weight control/maintenance. The focus of this dissertation was two-fold, 1) to evaluate the effectiveness of increased protein intake for promoting weight loss and improving body composition in a controlled weight loss study, and 2) to evaluate the use of the practice of "eating more protein" to prevent weight gain among midlife women. Additionally, the activity of the Renin-Angiotensin system (RAS) was evaluated to assess related metabolic effects with protein intake. The first study described the effect of three weight loss diets on body composition, blood pressure, and RAS metabolites. Midlife participants were randomly assigned a control diet (15% protein), a mixed protein diet (30% protein), and a whey protein diet (15% mixed, 15% whey) condition for a 5 month period. Total body weight and fat loss between groups was not significantly different, but a trend toward greater body weight and fat loss was observed with the whey protein diet. No differences in RAS metabolites were observed between diets, but a statistically significant decrease in systolic blood pressure was observed with the whey protein diet. These results confirm that reduction of energy intake is the primary effective step in weight loss, but secondary effects of regional fat loss and decreased blood pressure may be achieved with a high protein diet containing whey protein. The second study described survey results of a national panel of midlife women regarding weight maintenance practices and weight self-efficacy. In this cross-sectional survey, "eating more protein" was identified as the fourth most common practice used to prevent weight gain. Self-reported weight loss over 2 years was associated with reporting the use of the eating more protein practice. Although those who gained and lost weight reported similar weight maintenance practices, those who lost weight had significantly higher Weight Efficacy Lifestyle scores than those who gained weight. Educating individuals on the best use of protein to encourage successful weight maintenance may enhance the results. In conclusion, while many metabolic effects have been identified with increased protein intake, the best use practices for weight maintenance and weight loss continue to be a significant research topic. Increased protein intake has been associated with increased satiety and insulin sensitivity, and decreased blood pressure. Whey protein intake in a high protein weight loss plan may further result in regional fat loss and decreased blood pressure, but the specific mechanisms have not been determined. Among a national sample, midlife women reported eating more protein to maintain weight, and high self-efficacy was associated with successful weight maintenance.
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    Effects of the combination of protein and fiber in whole foods on satiety, blood glucose response and food intake in humans
    (2014-06) Bonnema, Angela
    The obesity epidemic continues worldwide and is considered a major health concern. A shift back to consumption of whole foods may be an important part of the solution in the current rising obesity trend. Both protein and fiber have satiating properties, but few studies have examined whether their impact on different biological mechanisms work additively to more strongly enhance the overall satiety potential of a meal. In the first study, the objective was to determine the effects of a high protein meal (beef) compared to a moderate protein, high fiber meal (beans) on subjective appetite and energy intake at a subsequent meal. We hypothesized that a moderate protein, high fiber meal containing beans would be as satiating as a high protein meal containing beef. Twenty-eight adults, 14 men and 14 women participated in this randomized, controlled study in which subjects consumed two test lunches including a "meatloaf" made from either beef or beans. The primary outcome was to observe satiety ratings using visual analogue scales to assess hunger, satiety, fullness, and prospective food intake. Secondary outcomes included: food intake at the subsequent meal offered in the form of snacks, gastrointestinal tolerance, and palatability of the meals. No difference between the beef and bean was observed for appetite ratings over 3 hours, food intake at the subsequent meal, or sum of GI score. Gas and bloating were reported more often after the bean meal than the beef meal. The conclusion to this first study was that a beef-based meal with high protein and a bean-based meal with moderate protein and high fiber produced similar satiety, while the bean-based meal resulting in higher, yet moderate, gas and bloating.In the second study, we examined the effect of egg alone and in combination with whole grains compared to a refined ready-to-eat cereal on satiety and food intake in human subjects. We hypothesized that breakfast meals containing eggs, both high protein with white toast and moderate protein with whole grain toast containing fiber, would result in increased satiety ratings compared to an isocaloric standard refined cereal breakfast. Forty-eight adults, 24 men and 24 women, participated in this randomized, cross-over study. We designed whole food diets, controlled for macronutrients. The primary outcome was to observe satiety ratings using visual analogue scales to assess hunger, satiety, fullness, and prospective food intake. Secondary outcomes included: post-prandial blood glucose response, food intake at the subsequent meal offered in the form of an ad libitum pizza lunch, gastrointestinal tolerance, and palatability of the meals. No difference was observed between the cereal and egg + whole grain toast breakfasts for AUC for all satiety ratings however the egg + white bread breakfast was significantly improved for all 4 satiety ratings. Lunch intake was significantly reduced in both egg breakfasts compared to the cereal breakfast. No difference was observed for the sum GI score between the egg + white toast, egg + whole grain toast and cereal breakfasts, however gas and bloating was significantly higher for the egg + white toast breakfast compared to the cereal meal. Food intake at the subsequent meal was reduced for both egg breakfasts compared to the cereal breakfast. The results from these studies support the hypothesis that protein and fiber contained within whole foods results in greater satiety than refined carbohydrate foods. Protein, with and without fiber, produced the greatest satiety outcomes suggesting that the incorporation of high protein foods into the diet, specifically for breakfast, may result in greater feelings of satiety that could lead to decreased food intake and weight loss over time.
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    Evaluate the Validity of Two Popular Food Oxidation Markers (Hexanal & Limonene Oxide) in the Dry State
    (2024-01) Wu, Xiaohan
    In research, it is common to use chemical indicators of oxidation to determine the oxidative state of a food. For example, hexanal formation/content is used to determine if lipids are oxidizing and limonene oxide (LO) serves a similar purpose in monitoring the oxidation of citrus oils. Recent research has shown that hexanal readily reacts with proteins and unpublished work suggests that limonene oxide also may react with proteins. Thus, these traditional indicators of oxidation may be inaccurate for foods that have higher protein levels or very reactive protein. β-Lactoglobulin (BLG) is a major whey protein in bovine milk and is also a prominent protein utilized in the food industry. Additionally, the structure and amino acid sequence of BLG is well characterized, and its molecular weight is appropriate for intact protein analysis. Therefore, BLG was selected as the model protein for this work. This research studied the covalent reactions between BLG and the two popular oxidation markers (hexanal and LO) in the dry state by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and to determine the influence of temperature, pH, and water activity on the reactions. Results show that limonene oxide could form covalent bonds with the BLG. Moreover, the reaction rate increases with the increase of storage time, which could make limonene oxide inaccurate as an oxidation marker in food containing high levels of proteins. For hexanal, the increase of the protein adduct is not very significant during storage. Therefore, hexanal still can be considered as a usable oxidation marker for dry foods.This research can help the food industry understand the validity of the two popular oxidation markers, choose better methods for food oxidation analysis, and improve their quality control system.
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    Heterogeneous protein distribution during rapid and equilibrium freezing
    (2013-04) Twomey, Alan Michael
    Interactions between proteins and ice were studied in situ using FTIR and confocal Raman microspectroscopy under equilibrium and non-equilibrium conditions over a range of temperatures. During quasi-equilibrium freezing of aqueous solutions of dimethyl sulfoxide (DMSO) and bovine serum albumin, preferential exclusion of albumin and/or DMSO was observed. It was hypothesized that the albumin may be adsorbed onto the ice interface or entrapped in the ice phase. To investigate protein-ice interactions during freezing under non-equilibrium conditions, confocal Raman microspectroscopy was used to map the distribution of albumin and the cryoprotective agent trehalose. Microheterogeneity was found in the composition of the freeze-concentrated liquid phase that indicated that albumin was preferentially distributed near or at the boundary of the ice phase. The observed microheterogeneity did not occur under all freezing protocols, which suggests that the technique developed here could be used to develop freezing protocols that would reduce harmful protein-ice interactions.
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    The incorporation of nitrosocyanin copper binding loop into azurin.
    (2010-07) Schenewerk, Audrey Rose
    Metalloprotein design and engineering can be used to probe our understanding of active site structure and function. Loop-directed mutagenesis has been used in the metalloprotein field to change the copper binding loops from a number of members of the cupredoxin family into other protein scaffolds. We report the replacement of a ten amino acid loop that supports the copper binding site in the blue copper protein azurin with the red copper binding loop from the protein nitrosocyanin. Azurin is an electron transfer protein while the role of nitrosocyanin is unknown, yet believed to be catalytic. In addition to the loop, we added a carboxylic acid residue into the copper binding site which fully models the site of nitrosocyanin. Synthesis, expression, and UV-visible absorption and EPR spectra for this series of azurin variants will be reported.
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    Novel Methods to Monitor Nutrition Status and Determine Protein Needs in Clinical Populations
    (2017-07) Kuchnia, Adam
    Malnutrition and muscle loss in hospitalized patients is a significant problem. The ability to accurately identify the development of malnutrition and monitor changes in muscle at the bedside are essential to optimizing nutrition interventions throughout treatment course and hospitalization; however, there is currently a lack of valid bedside tools. Furthermore, the provision of adequate protein and amino acids is crucial for maintaining muscle and optimizing outcomes in clinical settings. Yet, the methodologies used to determine protein needs have significant limitations, and current recommendations for dietary protein intake in clinical populations are not supported by strong evidence. This dissertation project consists of a series of studies that explore novel approaches for evaluating lean tissue and muscle (as core components of nutritional status) at the bedside, and determining protein requirements in the clinical setting. In the first study, using a large and ethnically-diverse healthy population sample (NHANES 1999-2004), it was determined that ethnicity significantly influences the values of phase angle (PA) and impedance ratio (IR), two bioimpedance parameters currently being investigated as clinical markers. Based on the findings from this study, cut-points for PA and IR corresponding to low muscle mass defined by dual-energy X-ray absorptiometry (DXA) were established that can potentially serve as reference data for future clinical studies investigating the applications of PA and IR as markers of lean tissue and/or nutritional status. In the second study, it was determined that PA and IR could be used to assess low muscularity and predict clinical outcomes in a large sample of critically ill patients. PA and IR were moderately associated with muscle cross-sectional area (CSA) as determined by computed tomography (CT). Furthermore, PA and IR appeared to predict low CT-derived muscle CSA. In summary, PA and IR show promise in being able to aid in the identification of low muscularity and poor nutritional status in the ICU setting. In the third and ongoing study, a novel stable amino acid isotope multi-step feeding protocol is being employed to determine the protein intake required to prevent net protein loss (anabolic threshold) and to evaluate the relationship between protein intake and net protein synthesis (anabolic capacity) of individuals with head and neck cancer (HNC), following chemoradiation therapy. Moreover, a force-measuring ultrasound (US) device is being used to assess changes in muscle quantity and quality due to chemoradiation. There is a vital need to develop objective bedside methods capable of assessing muscle mass in hospitalized patients. Results from the described studies show the potential utility of bioimpedance and US to characterize changes in muscle mass in various clinical populations. Furthermore, as clinicians become better equipped at detecting changes in muscle, appropriate nutritional intervention is needed to stave off the loss of muscle. Amino acid tracer methods that can estimate whole body protein synthesis and breakdown are fundamental to the determination of more accurate protein and amino acid recommendations in clinical populations in order to improve patient outcomes.
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    Novel microfluidic technologies: toward a low-cost system for protein crystallization.
    (2009-12) Hattan, Paul J
    The three-dimensional structure of folded proteins is of enormous interest to the scientific community. The structure is best determined with x-ray diffraction through a protein crystal, but it has proven extremely difficult to grow crystals large enough for this process [1, 2]. Significant challenges faced by protein crystallographers include the inability to sufficiently control the crystallization environment and the scarcity of protein available [3]. Microfluidic devices, which allow ultra-precise fluid management and require significantly less reagent than traditional methods, constitute an ideal technology with which to overcome these crystallization challenges [4-7]. A microfluidic system has been designed to give a crystallographer precise management of the concentrations of several reagents (such as protein and a suitable precipitant salt) over time. To create components of the microfluidic system, two novel fabrication methods were developed: photopolymer mold making and three-dimensional plate tectonics. These methods are rapid, inexpensive, and do not require any special equipment. A novel micropump and channel network suitable for the crystallization system were successfully created using these techniques.
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    Nuclear Magnetic Resonance and Circular Dichroism Spectroscopy to study the folding state of artificial, primordial-like proteins
    (2024-07-25) Blascyk, Eli
    Proteins are the primary molecular machinery for all known life. All proteins are made from the standard 20 amino acids in accordance to the universal genetic code of life; however, prior to the last universal common ancestor, this code was not set in stone and had to evolve to include each of the amino acids. The Seelig lab has previously generated libraries of random proteins using reduced sets of amino acids in order to simulate possible early protein functionality. The reduced sets of amino acids were based on the consensus chronology of incorporation into the genetic code and consisted of 5, 9, 16, and 20 of the amino acids used in modern proteins. This research aimed to characterize the structures of ATP binding proteins from each mRNA display library, primarily focusing on the 5AA and 9AA alphabet libraries. HSQC NMR and CD spectroscopy were used to characterize the structural and folding state of these proteins. Measurements from these spectroscopy experiments reveal secondary structure characteristics of up to 45% alpha helicity for individual variants. Further characterization efforts for these proteins are ongoing, including additional NMR and CD experiments as well as attempts to crystallize the proteins for X-ray crystallography.
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    Nutrient digestion and nitrogen metabolism of dried fermentation biomass and various fractions of rumen microbes.
    (2012-08) Carpenter, Abigail Joy
    An experiment was performed to investigate if fermentation biomass (FB), a dried bacterial co-product derived from lysine production (Ajinomoto Heartland, Inc.) can be used as a protein source in ruminant diets. Eight dual-flow continuous culture fermenters were inoculated with rumen fluid and used during one experimental period consisting of a 7-d adaptation period followed by 3 sampling days. Substrate for the microbes was provided by one of two isonitrogenous diets, CON or DFB. In CON, soybean meal (SBM) provided 57% of total CP, and in DFB, SBM and FB provided 12 and 45% of total CP, respectively. CON contained 3% molasses, 16% ground corn, 13% grass hay, 48% corn silage, and 20% SBM on a DM basis. DFB contained 3% molasses, 18.4% ground corn, 13% grass hay, 50% corn silage, 8.5% SBM, and 6.7% FB. Fermenters were supplied with 75 g/d of DM divided into 8 equal portions. Anaerobic conditions were maintained by infusion of N2; pH was maintained between 5.8 and 6.8; and temperature was set at 39C. On sampling days, liquid and solid effluent were collected, combined, and homogenized to be used for chemical analysis and in vitro estimation of intestinal digestibility (ID). Treatment did not affect (P > 0.1) average, maximum, or minimum fermenter pH. There was no effect (P > 0.1) on apparent or true OM, NDF, or ADF digestibility (%) or total VFA (mM), although branched-chain VFA (mM) was higher (P = 0.01) in CON because isobutyrate (mol/100 mol) tended (P = 0.06) to increase with CON treatment. Source of N had no effect (P > 0.1) on total, dietary, or bacterial-N flows. Addition of FB decreased (P < 0.05) NH3-N flow from 0.4 to 0.2 g/d and tended to decrease (P =0.06) effluent NH3-N concentration from 17.1 to 9.7 mg/100 mL. His and Met flows increased (P < 0.05) from 0.48 to 0.53 and 0.18 to 0.20 g/d, respectively, when FB partially replaced SBM in the diet, but there were no effects (P > 0.1) on other AA or total AA flows. There was a trend (P = 0.08) in percent non-essential AA input (CON = 73.6% vs. DFB = 82.2%) in effluent; however, there was no effect (P > 0.1) on percent of essential AA or total AA input in effluent. Effluent from the DFB treatment was higher (P < 0.05) in ID than CON (CON = 70.4% vs. DFB = 79.6%). These results indicate that FB elicited a similar response in N metabolism and AA flows to SBM but had a higher estimated digestibility in the intestine, and has potential use as a protein source in ruminant diets. In a second experiment, rumen fluid was collected from 4 steers fed the same diet, and 3 populations of rumen microorganisms (liquid-associated bacteria (LAB), solid-associated bacteria (SAB), and liquid-associated protozoa (LAP)) were isolated by differential centrifugation. Intestinal digestibility of N (ID) was determined using the in vitro (pepsin and pancreatin) steps of the 3-step procedure of Calsamiglia and Stern (1995). Total-N content of each fraction was different (P < 0.05) with LAB having the highest N content (8.1 vs. 6.1 vs. 5.6% of DM for LAB, SAB, and LAP, respectively). Purine concentration was greatest (P < 0.05) in LAB compared with SAB and LAP (3.1 vs. 1.0 vs. 0.9 mg/g bacterial DM, respectively). Similarly, LAB had a higher (P < 0.05) purine:N ratio compared with SAB and LAP (37.4 vs. 12.3 vs. 11.1 mg/g of bacterial N, respectively). Total amino acids (AA) (wt/wt) varied (P < 0.05) with each treatment (35.9 vs. 29.8 vs. 27.72% for LAB, SAB, and LAP, respectively). Most individual AA (g/100 g of total AA and wt/wt) exhibited differences (P < 0.05) between the 3 populations. Essential AA content (g/100 g of total AA) was highest (P < 0.05) in LAB and lowest in LAP (47.15 vs. 46.02, respectively). Non-essential AA content was greatest (P < 0.05) in LAP followed by SAB and LAB (53.3 vs. 52.3 vs. 51.8 g/100 g total AA, respectively). LAB was highest (P < 0.05) in ID followed by LAP and SAB (71.2% vs. 68.2% vs. 57.5% for LAB, LAP, and SAB, respectively). These results demonstrate that microbial fractions have differing digestibilities in the small intestine and suggest that ID of mixed rumen microbes can be influenced by the relative amount of each microbial population.
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    Patulin degradation by yeast protein extract
    (2014-05) Folger, Brian Charles
    The mycotoxin patulin, produced by a number of fungi, most prominently Penicillium expansum, has proven problematic for the apple industry due to contamination of apple juice and apple cider. Presently, techniques to control patulin accumulation have proven increasingly ineffective due to the presence of antifungal resistant strains of mold, stability of patulin during thermal processing, and conflicting data on the efficacy of other treatments. However, fermented apple products such as hard ciders and apple cider vinegars are devoid of patulin. Fermentation with yeast resulted in complete degradation of patulin, possibly due to enzymatic degradation by yeast enzymes. Patulin has also been shown to be susceptible to adduct formation with free thiol containing molecules such as glutathione, which is naturally present in yeast cells. Limited studies have also looked at patulin adsorptivity onto the call walls of yeast. Degradation of patulin is, therefore, hypothesized to be caused by multiple mechanisms mainly caused by yeast proteins/enzymes.To assess the loss of patulin by protein extracted from yeast (Rhodosporidium kratochvilovae strain 62-121), patulin extraction methods were compared to determine the optimal method for patulin extraction from protein rich environments. The effect of boiling to halt any possible enzymatic degradation on total patulin loss was assessed by comparing patulin recovery to that of samples placed on ice after the assay. Yeast growth was optimized for the production of patulin-degrading protein extracts by surveying days of growth and subsequent storage at 4°C. Additionally, free thiol group reactivity with patulin was assessed upon incubation with protein extract, cysteine, and glutathione. Liquid chromatography and mass spectrometry (LC/MS) was used to detect patulin degradation products. Potential enzymatic activity was assessed by comparing the degradation activity of different protein extracts from yeast. Finally, patulin loss due to adsorption to inactivated yeast cell walls was determined. The use of acid and salt to precipitate the protein before patulin extraction resulted in the best patulin recovery from protein rich media, and an additional extraction following a modified AOAC method allowed for removal of excess salt without sacrificing patulin recovery. The use of boiling to denature the protein after the assay resulted in 10% higher patulin loss than when the samples were placed on ice, presumably due to adduct formation with thiol groups. Growing yeast for 6 days at room temperature was deemed adequate to obtain optimal patulin degradation; and subsequent incubation of the yeast at 4°C did not impair the patulin degradation activity. Yeast protein extracts were found to be inconsistent with respect to patulin degradation activity, nevertheless patulin degradation activity (up to 100% patulin) was observed in several batches. Patulin incubated with cysteine showed signs of free thiol blockage in both samples of protein extract and pure cysteine. Patulin incubated with glutathione was degraded at both pH 7 and 3.7, and one patulin-glutathione adduct (462 m/z) was identified via LC/MS. Lyophilized yeast cells demonstrated patulin adsorption capabilities after incubation at 30°C for 20 min. Observed results confirm that patulin can be degraded by the protein extract from yeast. The exact mechanism of patulin degradation by protein extracts remains unclear, yet it appears to be either enzymatic or chemical through thiol adduct formation. Our results indicated that the mechanism is a combination of the two. This research offers insight into possible patulin degradation mechanisms, and can give direction in applying this new method of patulin control in an industrial setting.
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    A Substitute for Dystrophin: Why Utrophin Fails
    (2017) Ostgaard, Cyrina
    Each year 1 in 3500 males in the United States are born with Muscular Dystrophy (MD). In serious cases this disease is marked by heavily atrophied musculature, mental impairment, cardiomyopathy, and a shortened lifespan (~20 years). Currently, there is no cure for this debilitating disease and treatment options remain abysmal. Knowing how utrophin fails to replace dystrophin has potential for introducing factors that could fix this failure. That is part of the future of where this project is headed in terms of drug design and possible implications that could be had for disease treatment. This is one of the first projects that looks into the thermodynamics and molecular dynamics of these proteins to find how they function as little is still known about the way in which dystrophin transduces force and what role utrophin plays.
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    Utilization of oil crop residues for enhanced algae based production of lipids, polyunsaturated fatty acids, and protein
    (2013-12) Wang, Zhen
    Microalgae are capable of synthesizing many high value compounds, such as lipids, polyunsaturated fatty acids (PUFAs), and proteins, and therefore considered potential feedstock for production of biodiesel, nutraceuticals, and animal feeds. The synthesis and accumulation of lipids, PUFAs, and proteins are influenced by such factors as microalgae strain, trophic conversion mode of growth, nutrient supply, and light and temperature conditions. Regulation of these factors must take cost issue into account. The goal of this research was to investigate the use of oil crop biomass residue (OCBR), a low-cost material from oil extraction plants, to cultivate specific algae strains for targeted production of lipids, PUFAs, and proteins. The specific objectives of present thesis were: 1) to optimize the conditions for acid hydrolysis of OCBR for best yield and desirable profile of nutrients; 2) to evaluate microalgae growth on different OCBR media; 3) to characterize the chemical compositions especially the lipid, protein and EPA contents in the harvested algal biomass as affected by culture media; 4) to study the effect of temperature and growth phase on algae EPA synthesis.The one-factor-at-a-time experimental design was used to optimize the acid hydrolysis conditions based on the key nutrient level including the total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (COD) and ammonia nitrogen (NH4-N) in the hydrolysates. The optimal conditions were found to be using 3 % sulfuric acid and hydrolyzing residues at 90 °C for 20 hrs. The hydrolysates (OCBR media) produced under the optimal conditions were used to cultivate two algae strains, namely UM258 and UM268. The results from 5 days of cultivation showed that the best OCBR media supported faster algae growth than artificial media, with maximal algal biomass yield of 2.7 g/L and 3 g/L, respectively. Moreover, the total lipids after 5 days cultivation for UM258 and UM268 were 54 % and 35%, respectively. The OCBR also promoted protein accumulation in UM 268 compared with artificial media. Temperature-time interaction-effect on EPA synthesis was observed. Temperature of 20 °C and time of longer than 6 days of cultivation (after algae reach stationary phase), were found optimal for EPA accumulation for UM258 with EPA reaching 18% in total fatty acids. The results suggest that OCBR media are an excellent alternative for algae growth and have great potential for large scale productions of algae based ingredients for biodiesel, high value foods and animal feeds.