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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
131

Mutational Analysis and Characterization of Microbial Pesticides Isolated from Bacillus Thuringiensis

McNeil, Betina C. January 2011 (has links)
No description available.
132

Potential sources for the large scale production of human protein C

Morcol, Tulin 10 October 2005 (has links)
The vitamin K-dependent family of proteins (VKDs) include prothrombin, factors VII, IX, and X, and protein C (hPC) is synthesized in the liver and act to maintains normal hemostasis. such as properly regulated clotting. An imbalance of any of these pro- or anti-clotting proteins result in hemophilia or disseminated intravascular clotting diseases. Therefore, these proteins have a significant therapeutic value. Many of these proteins are not available in sufficient quantity due to the trace amounts found in plasma and limitations encountered with downstream recovery. Protein C, a major regulatory protein of thrombosis and hemostasis, has a potent anticoagulant activity and can be used as an anti-thrombotic agent. The technology for isolating hPC from human plasma is challenged by; (1) its low concentration in plasma, (2) the limited availability of plasma, (3) similar physicochemical characteristics among VKD plasma proteases, and (4) the risk of transmitting viruses such as the human immunodeficiency virus (HIV). This work focuses on the isolation of protein C from alternative sources for the large-scale production and downstream recovery of highly purified and biologically active hPC. The partial characterization of the protein with respect to post-translational modifications which are essential for functionally active, was also evaluated. Several studies were undertaken: 1. Cohn Fraction IV-I, an off-line discard stream during traditional plasma fractionation process is introduced as an affordable starting material for the large-scale production of hPC. More than 90 percent of the total protein C antigen detected in the various Cohn fractions was found to reside in fraction IV-I. The protein C isolated from Cohn IV-I paste using a metal-dependent monoclonal antibody to hPC was found to be biologically active. 2. Recombinant production of hPC in the milk of transgenic pigs, achieved by targeting the synthesis of the protein to the mammary gland, is presented as a model bioreactor system for the synthesis and downstream recovery of complex human proteins. Two major populations of biologically active recombinant hPC (rhPC) were detected and immunopurified by employing conformation specific metal-dependent monoclonal antibodies in the immunopurification process. A high performance thin layer chromatography method was also developed for the detection of total carbohydrate compositions in protein C. / Ph. D.
133

Protein Engineering for Biomedicine and Beyond

McCord, Jennifer Phipps 28 June 2019 (has links)
Many applications in biomedicine, research, and industry require recognition agents with specificity and selectivity for their target. Protein engineering enables the design of scaffolds that can bind targets of interest while increasing their stability, and expanding the scope of applications in which these scaffolds will be useful. Repeat proteins are instrumental in a wide variety of biological processes, including the recognition of pathogen-associated molecular patterns by the immune system. A number of successes using alternative immune system repeat protein scaffolds have expanded the scope of recognition agents available for targeting glycans and glycoproteins in particular. We have analyzed the innate immune genes of a freshwater polyp and found that they contained particularly long contiguous domains with high sequence similarity between repeats in these domains. We undertook statistical design to create a binding protein based on the H. magnipapillata innate immune TPR proteins. My second research project focused on creating a protein to bind cellulose, as it is the most abundant and inexpensive source of biomass and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept lignocellulosic fuels from competing commercially with starch-based biofuels. In recent years a strategy to protect processing enzymes with synergistic proteins emerged to reduce the amount of enzyme necessary for lignocellulosic biofuel production. Simultaneously, protein engineering approaches have been developed to optimize proteins for function and stability enabling the use of proteins under non-native conditions and the unique conditions required for any necessary application. We designed a consensus protein based on the carbohydrate-binding protein domain CBM1 that will bind to cellulosic materials. The resulting designed protein is a stable monomeric protein that binds to both microcrystalline cellulose and amorphous regenerated cellulose thin films. By studying small changes to the binding site, we can better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications such as enhancing the saccharification of lignocellulosic feedstock for biofuel production. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal scaffolds for tissue engineering and wound healing. However, the extraction process leads to protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Recombinant keratin biomaterials are free from these disadvantages, while heterologous expression of these proteins allows us to manipulate the primary sequence. We endeavored to add an RGD sequence to facilitate cell adhesion to the recombinant keratin proteins, to demonstrate an example of useful sequence modification. / Doctor of Philosophy / Many applications in medicine and research require molecular sensors that bind their target tightly and selectively, even in complex mixtures. Mammalian antibodies are the best-studied examples of these sensors, but problems with the stability, expense, and selectivity of these antibodies have led to the development of alternatives. In the search for better sensors, repeat proteins have emerged as one promising class, as repeat proteins are relatively simple to design while being able to bind specifically and selectively to their targets. However, a drawback of commonly used designed repeat proteins is that their targets are typically restricted to proteins, while many targets of biomedical interest are sugars, such as those that are responsible for blood types. Repeat proteins from the immune system, on the other hand, bind targets of many different types. We looked at the unusual immune system of a freshwater polyp as inspiration to design a new repeat protein to recognize nonprotein targets. My second research project focused on binding cellulose, as it is the most abundant and inexpensive source of biological matter and therefore is widely considered a possible source for liquid fuel. However, processing costs have kept cellulose-based fuels from competing commercially with biofuel made from corn and other starchy plants. One strategy to lower costs relies on using helper proteins to reduce the amount of enzyme needed to break down the cellulose, as enzymes are the most expensive part of processing. We designed such a protein for this function to be more stable than natural proteins currently used. The resulting designed protein binds to multiple cellulose structures. Designing a protein from scratch also allows us to study small changes to the binding site, allowing us to better understand how these proteins bind to different cellulose-based materials in nature and how to apply their use to industrial applications. Biomaterials made from natural human hair keratin have mechanical and biochemical properties that make them ideal for tissue engineering and wound healing applications. However, the process by which these proteins are extracted from hair leads to some protein degradation and brings with it byproducts from hair, which can cause unfavorable immune responses. Making these proteins synthetically allows us to have pure starting material, and lets us add new features to the proteins, which translates into materials better tailored for their applications. We discuss here one example, in which we added a cell-binding motif to a keratin protein sequence.
134

Combinatorial protein engineering to identify improved CRISPR activators

Giddins, Marla Jane January 2025 (has links)
Laboratory-engineered proteins such as high-fidelity DNA polymerases, CRISPR base and primeeditors, and chimeric antigen receptors have transformed our ability to probe and manipulate biological systems. To craft these powerful tools, researchers fuse multiple domains into novel chimeras intended to retain the functional properties of their constituent parts. Although this approach has produced a number of important technologies, its low-throughout nature and high costs thwart efforts to explore complex combinatorial landscapes and limit our grasp on the “rules” governing synthetic protein assembly (e.g., which domains work best together, which domain orders are optimal, benefits of fusing multiple copies of the same domain, etc.). Previous state-of-the-art CRISPR activators, including the tripartite activator, VP64-P65- RTA (VPR) and the Synergistic Activation Mediator (SAM), have established the benefit of combining multiple activation domains (ADs) into a single complex for improved transcriptional modulation. While VPR and SAM have proven relatively successful in both in vitro and in vivo applications, neither activator shows uniform activity across targets and cell types. Furthermore, reports that these tools produce toxicity within cellular systems limit their utility in broad-ranging applications. To probe a vast combinatorial landscape of multi-domain CRISPR activators while bypassing the arduous task of generating each construct one one-by-one, we developed a strategy for constructing large combinatorial libraries of protein variants en masse and used this method to functionally evaluate a library of >15,000 CRISPR activators. Importantly, we conduct our screen on multiple target genes to identify tools with consistent performance across the genome. Our findings bring to light a critical yet often overlooked feature of CRISPR activators: toxicity. This work not only highlights the prevalence of this problem but also elucidates several biological factors that contribute to it. Our observation that many high-performing activators elicited minimal effects on cell fitness challenges the notion that toxicity is an inevitable byproduct of a potent activation – and suggests that this model greatly oversimplifies the nuanced relationship between these traits. We also explored how the biochemical properties of ADs (e.g., hydrophobicity and intrinsic disorder) and their combinatorial interactions drive activator performance. Finally, we identified two potent activators, MHV and MMH, that show enhanced activity across diverse targets and cell types over one of the gold-standard CRISPR activators, SAM. Our results underscore the power of high-throughput techniques for both improving our understanding of complex protein assemblies and identifying more powerful tools.
135

Stereoselective production of dimethyl-substituted carbapenams via engineered carbapenem biosynthesis enzymes

Hamed, Refaat B., Henry, L., Claridge, T.D.W., Schofield, C. 2016 December 1928 (has links)
Yes / Stereoselective biocatalysis by crotonase superfamily enzymes is exemplified by use of engineered 5-carboxymethylproline synthases (CMPSs) for preparation of functionalized 5-carboxymethylproline (5-CMP) derivatives methylated at two positions (i.e. C2/C6, C3/C6 and C5/C6), including products with a quaternary centre, from appropriately-substituted-amino acid aldehydes and C-2 epimeric methylmalonyl-CoA. The enzymatically-produced disubstituted 5-CMPs were converted by carbapenam synthetase into methylated bicyclic Β-lactams, which manifest improved hydrolytic stability compared to the unsubstituted carbapenams. The results highlight the use of modi-fied carbapenem biosynthesis enzymes for production of new carbapenams with improved properties. / Medical Research Council, Biotechnology and Biological Sciences Research Council (BB/L000121/1)
136

High-throughput screening enabled advances in protein engineering

Kratz, Alexander Franz January 2024 (has links)
Nature has produced a dazzling array of proteins which perform useful and interesting functions. Over the last 50 years, biologists have begun to re-engineer these tiny machines, either to perform new functions, or to perform their functions more efficiently. However, protein engineering suffers from the massive scale of the search space. To improve the field’s ability to understand and engineer proteins, we present improvements both to generating and understanding large protein-function data sets. We apply these approaches to two tasks, generating data sets that measure the activity of tens of thousands of protein variants, and producing two novel CRISPR activators and an improved machine learning model for designing protein using DMS data as input data. In the first task, we engineer proteins at the level of domains, recombining trans-activation domains to generate improved CRISPR activators. By analyzing proteins at the level of domains, we simplify the protein engineering task into a smaller combinatorial problem. CRISPRa tools enable biologists to activate transcription at arbitrary locations using an easily retargetable CRISPR guide. In addition to producing two novel CRISPRa tools which outperform the current state-of-the-art, we perform what we believe to be the first systematic evaluation of the toxicity of CRISPRa tools in cells. We also perform a detailed analysis of the ways in which trans- activation domains interact in a multi-domain tool, and the impact of these interactions on both gene activation strength and toxicity. Our second protein engineering project approaches the problem at the level of individual amino acids. One target is a chaperone protein, DNAJB6, which our lab previously uncovered as a rescuer of toxicity for multiple neuro-degenerative proteins28. We use error-prone PCR to generate a library of over 30,000 compound mutants, which we screen using a yeast-based assay for their ability to rescue toxicity associated with an aggregation prone protein, FUS. We also engineer GFP, which has many existing deep mutational scanning (DMS) datasets. To engineer these proteins, we develop a machine learning model, OptiProt. Optiprot is trained on DMS data to approximate the sequence-to-score landscape and then perform machine- learning directed evolution (MLDE), designing proteins that meet user-defined criteria. We test OptiProt’s ability to learn and generalize from our DNAJB6 and GFP DMS datasets by adding increasingly difficult constraints and asking it to solve them. OptiProt was able to design proteins which improved on the best variants within the DMS data as well as integrate up to 50 mutations without breaking the functionality of the wildtype protein. Finally, we task OptiProt with difficult challenges such as compensating for a loss-of-function mutant or replacing every instance of a certain amino acid.
137

Investigation of the interactions between the bacterial homologue to actin, and the chaperone GroEL/ES through a combination of protein engineering and spectroscopy / Undersökning av interaktionerna mellan MreB, den bakteriella homologen till aktin, och chaperonet GroEL/ES genom en kombination av protein engineering och spektroskopi

Blom, Lillemor January 2008 (has links)
Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.
138

Protein surface charge of trypsinogen changes its activation pattern

Buettner, Karin, Kreisig, Thomas, Sträter, Norbert, Züchner, Thole 21 January 2015 (has links) (PDF)
Background: Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction. Results: Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme. Conclusion: The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.
139

Extending chemical complemenation to bacteria and furthering nuclear receptor based protein engineering and drug discovery

Johnson, Kenyetta Alicia 18 May 2009 (has links)
Nuclear receptors (NRs) are modular ligand-activated transcription factors that control a broad range of physiological processes by regulating the expression of essential genes involved in cell physiology, differentiation, and metabolism. These receptors are implicated in a number of diseases and due to their profound role in development and disease progression and their modularity, much emphasis is being put forth into nuclear receptor based drug discovery and engineering these receptors to bind novel small molecules Chemical Complementation (CC) is a yeast three-hybrid genetic selection system that was developed to aid in the discovery of these engineered receptors by linking the survival of a yeast cell to a small molecules ability to activate the receptor. Due to several advantages, to include faster growth times and higher transformation efficiencies, we have attempted to extend chemical complementation from yeast to E. coli. The bacterial chemical complementation system (BCC) was designed, based on a bacterial two hybrid system, to parallel yeast CC system. However, bacterial chemical complementation did not produce ligand dependent activation due to heterologous protein expression. In a second project designed to further NR based protein engineering and drug discovery, CC was used to evaluate a library of charge reversal variants rationally designed to gain a better understanding of nuclear receptor function and structure and to produce orthogonal ligand receptor pairs. A library of retinoic acid receptor (RARα) variants were developed based on five residues in the binding pocket known to stabilize the natural negatively charged ligand, all-trans retinoic acid (atRA). We altered the binding selectivity of the receptor to bind positively charged retinoid ligands. We were able to engineer two triple variants capable of activating with the positively charged retinoid but not the natural atRA ligand, however they do not activate as well as RARα wild-type does with atRA. In a third project we characterized covalently linked tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. Several dual inhibiting compounds were found to decrease the proliferation of ER positive breast cancer cells better than tamoxifen alone, the HDACi alone, or noncovalently linked HDACi and tamoxifen.
140

A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

Then, Johannes, Wei, Ren, Oeser, Thorsten, Gerdts, André, Schmidt, Juliane, Barth, Markus, Zimmermann, Wolfgang 23 June 2016 (has links) (PDF)
Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7°C (wild-type TfCut2: 69.8 °C) and its half-inactivation temperature to 84.6 °C (TfCut2: 67.3 °C). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 °C compared to 65 and 70 °C of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/-0.1%) at 70 °C after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.

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