Dissertations / Theses: 'Protein engineering'

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Sarkar, Mohosin M. "Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776.

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Taylor, M. J. "Protein engineering of staphylococcal protein A." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373965.

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Schymkowitz, Joost Wilhelm Hendrik. "Protein engineering studies on cell-cycle regulatory proteins." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621312.

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Wang, Hua. "Control of protein-surface, protein-protein, and cell-matrix interactions for biomaterials as tissue engineering scaffolds /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9894.

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McCord, Jennifer Phipps. "Protein Engineering for Biomedicine and Beyond." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/90787.

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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.

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Wilsher, Julie Ann. "Protein engineering of chymosin." Thesis, Birkbeck (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300804.

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Hao, Jijun. "Protein engineering of aldolases." Thesis, University of Leeds, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400182.

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Popplewell, Andrew George. "Protein engineering of protein-A from Staphylococcus aureus." Thesis, University of Southampton, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316403.

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Sun, Young Joo. "Engineering PDZ domain specificity." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6865.

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PSD-95/Dlg/ZO-1 (PDZ) domain - PDZ binding motif (PBM) interactions have been one of the most well studied protein-protein interaction systems through biochemical, biophysical and high-throughput screening (HTS) strategies. This has allowed us to understand the mechanism of individual PDZ-PBM interactions and the re-engineering of PBMs to bind tighter or to gain or lose certain specificity. However, there are several thousand native PDZ domains whose biological ligands remain unknown. Because of the low sequence identity among PDZ domain homologues, promiscuous binding profiles (defined as a PDZ domain that can accommodate a set of PBMs or a PBM that can be recognized by many PDZ domains), and context-dependent interaction mechanism, we have an inadequate understanding of the general molecular mechanisms that determine the PDZ-PBM specificity. Therefore, predicting PDZ specificity has been elusive. In addition, no de novo PBM ligand or artificial non-native PDZ domain have been successfully designed. This reflects the general challenges in understanding the general principles of PDZ-ligand interactions, namely that they are context-dependent, exhibit weak binding affinity, narrow binding energy range, and larger interaction surface than other protein-ligand interactions. Together, PDZ domains make good model systems to investigate the fundamental principles of protein-protein interactions with a wide spectrum of biomedical implications. My studies suggest that understanding PBM specificity with the set of structural positions forming the binding pocket can connect sequence, structure and function of a PDZ domain in a general context. They also suggest that this way of understanding the specificity will shed light on prediction and engineering of specificity rationally. Structural analysis on most of the available PDZ domain structures was established to support the principle (Chapter I). The principle was tested against two different types of PBM; C-terminal PBM (Chapter II) and internal PBM (Chapter III), and shown to support better understanding and design of PDZ domain specificity. We further applied the principle to design de novo PDZ domains, and the preliminary data hints that it is optimistic to engineer PDZ domain specificity (Appendix A and B).

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Xu, Ping. "Sensing and analyzing unfolded protein response during heterologous protein production :." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 205 p, 2008. http://proquest.umi.com/pqdweb?did=1555621341&sid=2&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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Maurus, Robert. "Protein engineering studies of myoglobin." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34587.pdf.

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He, Shiping. "Protein engineering of pea plastocyanin." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295349.

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Higgins, Jonathan M. G. "Protein engineering of human properdin." Thesis, University of Oxford, 1994. http://ora.ox.ac.uk/objects/uuid:80236e86-789e-4028-aad0-e72223f7645a.

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Properdin is a serum glycoprotein that upregulates the alternative pathway of complement by stabilizing the C3bBb complex. It also binds sulphated glycoconjugates, such as sulphatide, in vitro. Properdin is composed of cyclic dimers, trimers and tetramers of a 53 kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately sixty amino acids. The sixth TSR of properdin contains an insertion of approximately 30 amino acids which corresponds to the position of an intron in the human properdin gene. In order to identify the regions of properdin important for function, human properdin, and mutant forms each lacking a single TSR, were expressed in Chinese Hamster Ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of the normal and altered forms of properdin were investigated. Wild type recombinant properdin is similar to properdin purified from plasma in size, immunoreactivity, N-terminal sequence, possession of N-linked sugar, oligomerization (as determined by electron microscopy and gel exclusion chromatography), and functional activity in an alternative pathway haemolytic assay, and in C3b and sulphatide binding assays. Properdin "nicked" in TSR5 is unable to bind C3b, while retaining its overall structure and its ability to bind sulphatide. The removal of TSRS prevents C3b and sulphatide binding. Properdin lacking TSR4 is unable to stabilize the C3bBb complex, but is able to bind C3b and sulphatide, and shows the presence of monomers and dimers in the electron microscope. Properdin without TSR3 is able to stabilize the C3bBb complex, to bind CSb and sulphatide, and forms dimers, trimers and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3bBb complex. Monoclonal antibodies which bind to the N-terminal region, TSR1, or TSR2 are able to inhibit properdin binding to CSb. A monoclonal antibody which binds TSR4 is able to inhibit properdin binding to sulphatide, but not to CSb. The results confirm that TSRs are folded as independent units. The N-terminal end and TSR5 of properdin are implicated in CSb binding. The vertices of properdin oligomers may be important for interaction with CSb. TSR4 may also be involved in stabilization of the C3bBb complex. The sulphatide binding site is distinct from the CSb binding site, but TSR5, which contains many basic residues, may be important for both activities.

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Thomas, Paul Graham. "Protein engineering of subtilisin BPN'." Thesis, Imperial College London, 1988. http://hdl.handle.net/10044/1/47274.

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Liu, David Victor. "Protein engineering for cancer therapy." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/73796.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The immunosuppressive effects of CD4⁺CD25⁺ regulatory T cells (Tregs) interfere with anti-tumor immune responses in cancer patients. In the first part of this work, we present a novel class of engineered Interleukin-2 (IL-2) analogues that antagonize the IL-2 receptor, for inhibiting Treg suppression. These antagonists are engineered for high affinity to the IL-2 receptor a subunit and low affinity to either the [beta] or [gamma] subunit, resulting in a signaling-deficient IL-2 analogue that sequesters the IL-2 receptor a subunit from wild type IL-2. Using this design, human and mouse IL-2 antagonists were generated with inhibition constants ranging from 200 pM to 5 nM in vitro. Genetic fusions with IgG2a Fc enhanced serum half-life up to 30 hours. In order to study the effects of IL-2 antagonism, Fc fragments with disrupted effector functions were used. Fc-antagonist fusions bound to but could not deplete peripheral Tregs. They downregulated CD25 on Tregs, but could not perturb Treg function in a syngenic tumor model, presumably due to the high sensitivity of the IL-2 receptor and a high threshold for antagonism in vivo. In the second part of this work, we present a novel multi-agent protein-based system for targeted siRNA delivery that provides potential advantages over other nanoparticle- and proteinbased delivery vehicles. In the first agent, the double stranded RNA binding domain (dsRBD) of human protein kinase R is used as an siRNA carrier, in fusion proteins that target epidermal growth factor receptor (EGFR). Targeted dsRBD proteins deliver large amounts of siRNA to endosomal compartments in an EGFR expressing cell line, but efficient gene silencing is limited by endosomal escape. The use of a second agent that contains the cholesterol dependent cytolysin, perfringolysin 0, enhances endosomal escape of siRNA. Targeted delivery of perfringolysin 0 induces gene silencing in a dose-dependent and EGFR-dependent manner. However, cytotoxicity of the cytolysin creates a narrow therapeutic window. Multiepitopic EGFR binders that induce EGFR clustering are explored as tools for enhancing gene silencing efficiency. Interestingly, they not only enhance gene silencing potency but also protect against toxicity from EGFR-targeted cytolysins, thus significantly widening the therapeutic window of this method.
by David Victor Liu.
Ph.D.

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Füllen, Georg Karl-Heinz. "Protein engineering and pattern recognition." Thesis, Massachusetts Institute of Technology, 1994. http://hdl.handle.net/1721.1/17354.

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Parker, Rachael N. "Protein Engineering for Biomedical Materials." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/77416.

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The inherent design freedom of protein engineering and recombinant protein production enables specific tailoring of protein structure, function, and properties. Two areas of research where protein engineering has allowed for many advances in biomedical materials include the design of novel protein scaffolds for molecular recognition, as well as the use of recombinant proteins for production of next generation biomaterials. The main focus of my dissertation was to develop new biomedical materials using protein engineering. Chapters three and four discuss the engineering of repeat proteins as bio-recognition modules for biomedical sensing and imaging. Chapter three provides an overview of the most recent advances in engineering of repeat proteins in the aforementioned field. Chapter four discusses my contribution to this field. We have designed a de novo repeat protein scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Innate immunity receptors have been described as pattern recognition receptors in that they recognize "global features" of a family of pathogens versus one specific antigen. In mammals, two main protein families of such receptors are: extracellular Toll-like receptors (TLRs) and cytoplasmic Nucletide-binding domain- and Leucine-rich Repeat-containing proteins (NLRs). NLRs are defined by their tripartite domain architecture that contains a C-terminal LRR (Leucine Rich Repeat) domain, the nucleotide-binding oligomerization (NACHT) domain, and the N-terminal effector domain. It is proposed that pathogen sensing in NLRs occurs through ligand binding by the LRR domain. Thus, we hypothesized that LRRs would be suitable for the design of alternative binding scaffolds for use in molecular recognition. The NOD protein family plays a very important role in innate immunity, and consequently serves as a promising scaffold for design of novel recognition motifs. However, engineering of de novo proteins based on the NOD family LRR domain has proven challenging due to problems arising from protein solubility and stability. Consensus sequence design is a protein design tool used to create novel proteins that capture sequence-structure relationships and interactions present in nature in order to create a stable protein scaffold. We implement a consensus sequence design approach to develop proteins based on the LRR domain of NLRs. Using a multiple sequence alignment we analyzed all individual LRRs found in mammalian NLRs. This design resulted in a consensus sequence protein containing two internal repeats and separate N- and C- capping repeats named CLRR2. Using biophysical characterization methods of size exclusion chromatography, circular dichroism, and fluorescence, CLRR2 was found to be a stable, monomeric, and cysteine free scaffold. Additionally, CLRR2, without any affinity maturation, displayed micromolar binding affinity for muramyl dipeptide (MDP), a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand. Furthermore, CLRR2 demonstrated selective recognition to the biologically active stereoisomer of MDP. Results of this study indicate that LRRs are indeed a useful scaffold for development of specific and selective proteins for molecular recognition, creating much potential for future engineering of alternative protein scaffolds for biomedical applications. My second research interest focused on the development of proteins for novel biomaterials. In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues, as well as participates in important regulatory functions such as cell migration and proliferation and protein signalling. The aforementioned properties along with keratins' inherent capacity for self-assembly poise it as a promising scaffold for regenerative medicine and tissue engineering applications. However, due to the extraction process used to obtain natural keratin proteins from natural sources, protein damage and formation of by-products that alter network self-assembly and bioactivity often occur as a result of the extensive processing conditions required. Furthermore, natural keratins require exogenous chemistry in order to modify their properties, which greatly limits sequence tunability. Recombinant keratin proteins have the potential to overcome the limitations associated with the use of natural keratins while also maintaining their desired structural and chemical characteristics. Thus, we have used recombinant DNA technology for the production of human hair keratins, keratin 31 (K31) and keratin 81 (K81). The production of recombinant human hair keratins resulted in isolated proteins of the correct sequence and molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. Proteins with no unwanted sequence truncations, deletions, or mutations indicate recombinant DNA technology can be used to reliably generate full length keratin proteins. This allows for consistent starting materials with no observable impurities or undesired by-products, which combats a major challenge associated with natural keratins. Additionally, recombinant keratins must maintain the intrinsic propensity for self-assembly found in natural keratins. To test the propensity for self-assembly, we implemented size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to characterize K31, K81, and an equimolar mixture of K31 and K81. The results of the recombinant protein characterization reveal novel homo-polymerization of K31 and K81, not previously reported, and formation of characteristic keratin fibers for the K31 and K81 mixture. Therefore, recombinant K31 and K81 retain the intrinsic biological activity (i.e. self-assembly) of natural keratin proteins. We have also conducted a comparative study of recombinant and extracted heteropolymer K31/K81. Through solution characterization and TEM analysis it was found that use of the recombinant heteropolymer allows for increased purity of starting material while also maintaining self-assembly properties necessary for functional use in biomaterials design. However, under the processing condition implemented, extracted keratins demonstrated increased efficiency of assembly. Through each study we conclude that recombinant keratin proteins provide a promising solution to overcome the challenges associated with natural protein materials and present an exceptional design platform for generation of new biomaterials for regenerative medicine and tissue engineering.
Ph. D.

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Volpe, Adua <1996&gt. "Protein engineering for drug transport." Master's Degree Thesis, Università Ca' Foscari Venezia, 2021. http://hdl.handle.net/10579/20470.

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Negli ultimi anni, le nanoparticelle sono emerse come nuovi vettori promettenti per la somministrazione di terapie e farmaci. Le molecole naturali, come le proteine, sono un ottimo sostituto dei polimeri sintetici comunemente impiegati nella sintesi di nanoparticelle grazie alla loro sicurezza, biodisponibilità, biocompatibilità, biodegradabilità e bassissima tossicità. Inoltre la loro sintesi è ecologica e non prevede l'utilizzo di sostanze chimiche tossiche. I sistemi di somministrazione più utilizzati sono quelli basati su proteine naturali, come l'albumina sierica umana. L'albumina sierica umana è la proteina più abbondante nel corpo umano con una lunga emivita. La sua struttura caratteristica consente il legame multiplo di sostanze terapeutiche sulla sua superficie, rendendo l'HSA la proteina più utilizzata e prominente per i sistemi di somministrazione di farmaci a base di proteine. È noto che l'HSA tende ad accumularsi all'interno del sito del tumore o nei tessuti infiammati a causa del trasporto mediato dal recettore. Lo scopo di questa tesi era cercare di ingegnerizzare l'albumina sierica umana per i sistemi di somministrazione di farmaci. Alcuni studi hanno rivelato che gli oligomeri di HSA, in un certo intervallo di dimensioni, sembrano molto promettenti come carrier per le terapie. Infatti, grazie alla loro dimensione specifica, l'assorbimento da parte del fegato e la distruzione da parte del sistema immunitario sono notevolmente ridotti o evitati. Questa tesi illustra la proteina impiegata (albumina sierica umana) e i metodi utilizzati nella generazione di oligomeri proteici, come la clonazione, la tecnica di assemblaggio di Gibson, l'elettroforesi su gel.

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Shakalli, Miriam Joan. "Applied Protein Engineering for Bacterial Biosensor and Protein Purification." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1450805485.

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Campbell, Sean Thomas. "Protein Engineering for Biochemical Interrogation and System Design." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/560940.

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Proteins are intimately involved in almost every cellular phenomenon, from life to death. Understanding the interactions of proteins with each other and other macromolecules and the ability to rationally redesign them to improve their activities or control their function are of considerable current interest. Split-protein methodologies provide an avenue for achieving many of these goals. Since the original discovery of conditionally activated split-ubiquitin, the field has grown exponentially to include the activities of over a dozen different proteins. The flexibility of the systems has resulted in their use across a wide spectrum, both literally and figuratively, to primarily screen, visualize and quantitate macromolecular interactions in a variety of biological systems. In another arena, there is significant interest the apoptosis-regulating proteins: the Bcl-2 family. These proteins are found in many cell types and control, through expression levels as well as other mechanisms, the apoptotic state of a protein as governed by intrinsic death signals generated from such sources as DNA damage and viral infection. The apoptotic function of these proteins are mainly governed by a single type of interaction: the helix:receptor binding of the BH3-Only helices to the anti-apoptotic receptor proteins. While this often promiscuous helix:receptor interaction has received much scrutiny, the nature of the anti-apoptotic binding pocket, especially with regard to the specific residues that govern the interaction, has been lacking. With the high sensitivity and rapid analysis platform afforded by the cell-free split-luciferase analysis methodology, we devised and carried out the first systematic and large scale alanine mutagenesis of all five major anti-apoptotic members of the Bcl-2 family, validated these results both with biophysical methods as well as correlation with previous studies. Our results help explain how different receptors can bind a wide range of helices and also uncovered details regarding binding that are not possible with structural or computational analysis alone. In a second area of research, we have utilized the interaction of BH3 helices and their receptors for designing small molecule controlled protein kinases and phosphatases. In this protein design area, BH3-Only helices were inserted using a knowledge based approach into particular loops within both a protein kinase and a protein phosphatase. The BH3-Only helix interaction with added receptors, such as Bcl-xL provided an allosteric switch for turning-off the activity of the helix-inserted enzymes. The activity of the enzymes could then be turned-on by the addition of a cell-permeable small molecule that is known to bind the receptor. This plug-and-play design was demonstrated to be successful for two very different enzyme classes and likely provides a general and tunable biological element for controlling the activity of one or more proteins and enzymes in a biochemical networks.

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Cummings, Chad S. "High Density Polymer Modification of Proteins Using Polymer - Based Protein Engineering." Research Showcase @ CMU, 2016. http://repository.cmu.edu/dissertations/692.

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Proteins and protein-based materials are used for a wide range of therapeutic, diagnostic, and biotechnological applications. Still, the inherent instability of proteins in non-native environments greatly limits the applications in which they are effective. In order to increase their utility, proteins are often modified, either biologically or chemically, to manipulate their bioactivity and stability profiles. In this work, covalent attachment of polymers to the enzyme chymotrypsin was used to predictably tailor protein bioactivity and stability. Specifically, atom transfer radical polymerization (ATRP) based polymer-based protein engineering (PBPE) was used to grow polymers directly from the surface of chymotrypsin. First, the temperature responsive polymers poly(N-isopropyl acrylamide) (pNIPAM), which has a lower critical solution temperature (LCST) and poly(dimethylamino propane sulfonate) (pDMAPS), which has an upper critical solution temperature (UCST), were separately grown from chymotrypsin. The temperature responsive properties of the polymers were conserved in the protein-polymer conjugates, and chymotrypsin bioactivity, productivity, and substrate specificity were predictably tailored at different temperatures depending on the structural organization of the polymers. Next, a dual block polymer-chymotrypsin conjugate was synthesized by growing poly(sulfobetaine methacrylamide) (pSBAm)-block-pNIPAm conjugates from the surface of chymotrypsin. The CT-pSBAm-b-pNIPAm conjugates showed temperature dependent kinetics, due to UCST or LCST driven polymer collapse at high and low temperature. Most interestingly, the dual block conjugates were dramatically more stable than native chymotrypsin to low pH. In order to further investigate the effect of polymer conjugation on chymotrypsin stability at low pH, four distinct and uniquely charged polymers were grown from the surface of chymotrypsin. With these new conjugates, we confirmed that chymotrypsin low pH stability was dependent on the chemical structure of polymers covalently attached to chymotrypsin. Indeed, positively charged polymers stabilized chymotrypsin to low pH, but negatively charged and amphiphilic polymers destabilized the enzyme. Lastly, after developing strategies for low pH stabilization, new protein-polymer conjugates with the chemical permeation enhancer 1-phenylpiperazine were designed to enable protein transport across the intestinal epithelium. Bovine serum albumin-poly(oligoethylene methacrylate)-block-poly(phenylpiperazine acrylamide) BSA-pOEGMA-b-pPPZ conjugates induced dose dependent increases in Caco-2 monolayer permeability and transported across an in vitro intestinal monolayer model with low cell toxicity.

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Alahuhta, M. (Markus). "Protein crystallography of triosephosphate isomerases: functional and protein engineering studies." Doctoral thesis, University of Oulu, 2008. http://urn.fi/urn:isbn:9789514287909.

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Abstract The aim of this PhD-study was to better understand the structure-function relationship of triosephosphate isomerase (TIM) and to use this expertise to change its substrate specificity. TIM is an important enzyme of the glycolytic pathway which catalyzes the interconversion of D-glyceraldehyde phosphate (D-GAP) and dihydroxyacetone phosphate (DHAP). Two main subjects are discussed: the engineering of monomeric TIM to create new substrate specificity and the structure-function relationship studies of the catalytically important mobile loop6. The starting point for the protein engineering project was the monomeric ml8bTIM, with an extended binding pocket between loop7 and loop8. Rational protein engineering efforts have resulted in a new variant called A-TIM that can competently bind wild type transition state analogues. A-TIM was also able to bind citrate, a compound that the wild type TIM does not bind. This A-TIM citrate complex structure is a good starting point for future protein engineering efforts. Based on the assumption that it would be beneficial for the monomeric forms of TIM to have loop6 closed permanently to increase the population of competent active sites, two point mutation variants, A178L and P168A were generated and characterized. The A178L-mutation was made to favor the closed conformation of loop6 through steric clashes in the open conformation. The P168A variant was made to stabilize the closed conformation of loop6 by removing strain. The A178L mutation induced some features of the closed conformation, but did not result in a closed conformation in the absence of ligands. Our structural studies also show that the P168A mutation does not favor the closed conformation either. However, the structures of the unliganded and liganded P168A variant, together with other known TIM structures show that the substrate binding first induces closure of loop7. This conformational switch subsequently forces loop6 to adopt its closed conformation. The protein engineering project was successful, but the efforts to find variants with a permanently closed loop6 did not fully succeed. In the context of this thesis a monomeric variant of TIM, with new binding properties, was created. Nevertheless, A-TIM still competently binds the inhibitors and transition state analogues of wild type TIM. Also, when combined, results discussed in the context of this thesis indicate that in wild type TIM the closure of loop7 after ligand binding is the initial step in the series of conformational changes that lead to the formation of the competent active site
Tiivistelmä Tämän väitöskirjatyön tarkoituksena oli oppia paremmin ymmärtämään trioosifosfaatti-isomeraasin (TIM) toimintamekanismeja sen rakenteen perusteella ja käyttää tätä tietämystä samaisen proteiinin muokkaamiseen uusiin tarkoituksiin. TIM on keskeinen entsyymi solun energian tuotannossa ja sen toiminta on välttämätöntä kaikille eliöille. Tämän vuoksi on tärkeää oppia ymmärtämään miten se saavuttaa tehokkaan reaktionopeutensa ja miksi se katalysoi vain D-glyseraldehydi-3-fosfaattia (D-GAP) ja dihydroksiasetonifosfaattia (DHAP). TIM:n toiminta mekanismien ymmärtämiseksi sen aminohapposekvenssiä muokattiin kahdesta kohtaa (P168A ja A178L) ja seuraukset todettiin mittaamalla tuotettujen proteiinien stabiilisuutta optisesti eri lämpötiloissa ja selvittämällä niiden kolmiulotteinen rakenne käyttäen röntgensädekristallografiaa. Mutaatioita tehtiin dimeeriseen villityypin TIM:in (wtTIM) ja jo aikaisemmin muokattuun monomeeriseen TIM:in (ml1TIM). Näiden mutaatioiden tarkoituksena oli suosia entsyymin aktiivista konformaatiota, jossa reaktion kannalta välttämätön vapaasti liikkuva peptidisilmukka numero 6 on suljetussa konformaatiossa. Monomeerisissä TIM:ssa peptidisilmukka numero 6:n ei ole välttämätöntä aueta. Tulokset mutaatiokokeista olivat osittain lupaavia. P168A-mutaatio lisäsi D-GAP:in sitoutumista, mutta rikkoi tärkeän mekanismin suljetussa, ligandia sitovassa, konformaatiossa. A178L-mutaatio aiheutti muutoksia avoimeen konformaatioon ja teki siitä suljettua konformaatiota muistuttavan jopa ilman ligandia, mutta samalla koko proteiini muuttui epävakaammaksi. Näistä kahdesta mutaatiosta A178L voisi olla hyödyllinen muokattujen TIM-versioiden ominaisuuksien parantamiseksi. Lisäksi yhdessä jo aikaisemmin julkaistujen yksityiskohtien kanssa nämä tulokset tekevät mahdolliseksi esittää tarkennusta siihen miten TIM toimii kun ligandi saapuu sen lähettyville. Tämän väitöskirjatyön yksi tavoite oli myös muokata edelleen monomeeristä TIM versiota (ml8bTIM), joka on suunniteltu siten, että se voi mahdollisesti sitoa uudenlaisia ligandeja. Tämä projekti vaati onnistuakseen 20 eri versiota ml8bTIM:n sekvenssistä ja noin 30 rakennetta. Uusia ligandeja sitova muoto (A-TIM) sitoi onnistuneesti sitraattia ja villityypin TIM:n inhibiittoreita. Erityisen lupaavaa oli, että A-TIM sitoi myös bromohydroksiasetonifosfaattia (BHAP), joka sitoutuu ainoastaan toimivaan aktiiviseen kohtaan. Nämä tulokset osoittavat, että A-TIM kykenee tarvittaessa katalysoimaan isomerisaatio reaktion uudenlaisille molekyyleille. Esimerkiksi katalysoimaan isomerisointireaktiota sokerianalogien tuotannossa

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Karlsson, Andreas. "Characterization and Engineering of Protein-Protein Interactions Involving PDZ Domains." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-312872.

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The work presented in this thesis has contributed with knowledge to several aspects of protein-protein interaction involving PDZ domains. A substantial amount of our proteome contains regions that are intrinsically disordered but fold upon ligand interaction. The mechanism by which disordered regions bind to their ligands is one important piece of the puzzle to understand why disorder is beneficial. A region in the PDZ domain of nNOS undergoes such a disorder-to-order transition to form a b-sheet in the binding pocket of its partner. By studying the kinetics of interaction, in combination with mutations that modulate the stability of the aforementioned region, we demonstrate that the binding mechanism consists of multiple steps in which the native binding interactions of the b-sheet are formed cooperatively after the rate-limiting transition state. These mechanistic aspects may be general for the binding reactions of intrinsically disordered protein regions, at least upon formation of β-sheets. The second part of this thesis deals with the engineering of proteins for increasing affinity in protein-protein interaction. Infection by high-risk human papillomavirus (hrHPV) can lead to cancer, and the viral E6 protein is an attractive drug target. E6 from hrHPV natively interacts with the well-characterized PDZ2 domain in SAP97, which we used as a scaffold to develop a high affinity bivalent binder of hrHPV E6. We initially increased PDZ2's affinity for E6 6-fold, but at the cost of decreased specificity. Attaching a helix that binds E6 at a distant site, increasing the affinity another14-fold, completed the design. The final work of this thesis investigates if binding studies conducted with isolated PDZ domains is representative of the full-length proteins they belong to. It has been suggested that ligand binding in PDZ domains can be influenced by factors such as adjacent domains and interactions outside of the binding pocket. We studied these aspects for the three PDZ domains of PSD-95 and found that they on the whole function in an independent manner with short peptides as ligands, but that interactions outside of the PDZ binding-pocket may be present. The representative length of the PDZ interaction partner should therefore be considered.

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Zhang, Xinyi. "PROTEIN ENGINEERING IN THE STUDY OF PROTEIN LABELING AND DEGRADATION." UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/93.

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Proteins are large macromolecules that play important roles in nature. With the development of modern molecular biology techniques, protein engineering has emerged as a useful tool and found many applications in areas ranging from food industry, environmental protection, to medical and life science. Biomimetic membrane incorporates biological elements, such as proteins, to form membranes that mimic the high specificity and conductance of natural biological membranes. For any application involving the usage of proteins, the first barrier is always the production of proteins with sufficient stability, and the incorporation of proteins into the artificial matrix. This thesis contains two major parts, the first part is focused on the development and testing of method to immobilize active enzymes. The second part is devoted to study the degradation of membrane proteins in E. coli cells. In the immobilization study, Pyrophosphatase (PpaC) was chose as a model enzyme. A dual functional tag consist of histidine and methionine has been developed, in which histidine is used for purification while methionine is metabolically replaced with azidohomoalanine (AHA) for immobilization. We found that the addition of the tag and the incorporation of AHA did not significantly impair the properties of proteins, and the histidine–AHA tag can facilitate protein purification, immobilization, and labeling. This tag is expected to be useful in general for many proteins. Degradation of soluble protein has been well characterized, but the membrane protein degradation process remains elusive. SsrA tag is a well-known recognition sequence for soluble protein degradation, which marks prematurely terminated protein products translated from damaged mRNA. SsrA tagged membrane proteins was found to be substrate of a cytosolic protease complex ClpXP, which mediated complete degradation.

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Forghani, Farnaz. "Protein engineering of bacteriophage Mu transposase." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60444.

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Bacteriophage Mu is an ideal system to study DNA transposition. The 70-KDa protein product of the phage early gene A, termed transposase, is absolutely required for transposition. Transposase binds specifically at sites located at both ends of the phage genome, termed attL and attR, and at an enhancer-like element at the left end of the genome, called IAS (internal activation sequence). It then nicks at these ends, and nicks a random target DNA sequence in a 5 base pair staggered fashion with 5$ sp prime$ extensions and promotes strand transfer between the Mu ends and the target DNA. The transposase protein can be roughly divided into three domains. The other activities of the protein have not been mapped even at the domain level. To further define the different functional domains of this complex enzyme, a series of insertion mutants at 8 different sites along the transposase protein were constructed using TAB linker mutagenesis. In this study, 1 and 2 TAB linkers were inserted into 8 HpaII sites in the Mu A gene, generating a set of 2 and 4 amino acid insertion mutants. Examination of these mutants for specific DNA-binding activity of transposase to the ends of the phage genome in vitro revealed temperature sensitive proteins. Transpositional activity of the mutant proteins revealed that the mutant proteins, which are temperature sensitive in specific DNA-binding activity, are deficient in transpositional activity.

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Hellinga, H. W. "Protein engineering of E. coli phosphofructokinase." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377195.

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Hughes, Paul Edward. "Protein engineering of human factor IX." Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306171.

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Gibson, Rosemary M. "Protein engineering of #Beta#-lactamase 1." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.256768.

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Hilyard, Katherine L. "Protein engineering of antibody combining sites." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291278.

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Messer, Neil Gavin. "Protein engineering of myosin light chains." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316757.

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Brannigan, James Anthony. "Strategies for protein engineering of subtilisin." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/47786.

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Lau, T. F. "Protein engineering of E. coli phosphofruktokinase." Thesis, Imperial College London, 1988. http://hdl.handle.net/10044/1/47149.

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Field, James Edward John. "Engineering protein cages with synthetic biology." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/45404.

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Nanotechnology has the potential to revolutionise every facet of human life. One particularly exciting branch of nanotechnology involves the construction of nanodevices using protein cages. Protein cages are spherically shaped structures with large internal cavities. The research described in this thesis was conducted with the aim of rationalising the design and fabrication of protein cage-based nanodevices. Protein-based nanodevices are typically constructed by re-engineering naturally occurring protein chassis (e.g. ferritin). To rationalise the process of chassis selection, an online registry of protein cages, rings and tubes was designed and populated by computationally mining the Protein Data Bank. The resulting registry was made publically available to the research community through the website – www.nanodevice.build. The functionality of protein cage-based nanodevices can be augmented by packaging inorganic nanoparticles inside their internal cavities. The methods currently used to achieve this typically involve exposure to harsh conditions, which can cause irreversible damage to the protein cage. To address this, a strategy for efficiently packaging inorganic nanoparticles into protein cages under mild conditions was formulated and tested. These experiments were conducted using gold nanoparticles and a number of different protein cages (e.g. Bfr, FtnH and FtnL). Cholangiocarcinoma (CCA) is a deadly liver cancer for which current treatment options are limited. Therefore a CCA-targeting protein cage-based nanodevice was designed, constructed and experimentally evaluated. CCA-targeting was achieved in the context of the CCA cell line TFK-1 using an anti-mesothelin antibody as a targeting agent. Collectively, these three outputs provide a rational framework for selecting a protein cage chassis, loading it with a pre-fabricated inorganic nanoparticle and targeting the resulting device to a particular cell-type. It is hoped that by leveraging these three tools, synthetic biologists will be able to engineer a new generation of nanodevices.

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Haji, Ruslan Khairunnisa Nabilah. "Protein hydrogels as tissue engineering scaffolds." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/protein-hydrogels-as-tissue-engineering-scaffolds(45ff4e72-49ea-46df-9e7b-b9113576c096).html.

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Hydrogels aim to mimic the natural living environment by entrapping large amount of water or biological fluids in their polymeric network. There has been growing interest in the development of peptide and protein hydrogels, due to their improved biocompatibility, biodegradability and biological properties in comparison to purely synthetic polymer hydrogels. Under the appropriate conditions, biomacromolecular protein hydrogels can self-assemble into ordered meso- to macroscopic supramolecules with better resulting networks that promote tissue development. The work presented here mainly focuses on producing protein hydrogels with controlled physical properties useful for tissue regeneration process and drug delivery applications. Hen egg white lysozyme (HEWL) hydrogels were studied in the presence of water and different reducing agents forming three HEWL systems including HEWL/water, HEWL/DTT and HEWL/TCEP gels. Strong, self-supporting HEWL gels were successfully prepared in the range of pH 2 to 7, using a temperature of 85°C. At pH 2, the protein denaturation in water was relatively slow resulting in a high percentage of turn structure (~50%) that promotes HEWL gelation after 3 days of heating. No lysozyme gelation in water was observed at pH 3, 4 and 7 even after 21 days of heating. A small quantity of DTT (~20 mM) was added to encourage lysozyme unfolding and HEWL/DTT samples formed gels at higher pH including at physiological pH. The pH 2 HEWL/water gel was found to be stronger but more brittle than pH 7 HEWL/DTT gel. It was observed there were some irregularities in the distribution of pH 2 fibrils (~7µm in length) that form large pore sizes within the network. The pH 7 sample contained shorter and stiff fibrils with repetitive polygon-shaped mesh network. The use of TCEP, which is a stronger reductant than DTT, led to the formation of self-supporting HEWL gels between pH 3.5 and 5.5. The highest storage modulus was observed at pH 5, which is related to the high β-sheet content of the sample (~45%). In addition, a promising strategy has been devised to form thermoresponsive HEWL hydrogels by synthesising and incorporating a small fraction of lysozyme-PNIPAAm bioconjugates into the major protein matrix. Results show the thermoresponsive nature of PNIPAAm was conferred to HEWL protein that exhibits higher storage stability in response to changing temperature.

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Kwan, Ann H. Y. "Protein design based on a PHD scaffold." Connect to full text, 2004. http://setis.library.usyd.edu.au/adt/public_html/adt-NU/public/adt-NU20041202.102526/index.html.

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Thesis (Ph. D.)--School of Molecular and Microbial Biosciences, Faculty of Science, University of Sydney, 2004.
Chapter headings on separately inserted unnumbered cream coloured leaves. Bibliography: leaves 122-135.

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Doolan, Kyle M. "Engineering and characterization of protein-protein interactions in prion disease and therapy." Thesis, University of Delaware, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=3730253.

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Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP^C, into a disease-associated conformation, PrP^Sc, that is β-sheet rich and can form amyloid deposits in the brain. PrP^Sc formation induces neuronal death and an invariably fatal neurodegenerative disease. The pathology of prion diseases is among the best understood of a group of neurodegenerative diseases that show similar features including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease because it is experimentally infectious. Samples containing PrP^Sc when introduced into a host bind to native PrP^C and promote conversion to PrP^Sc in a seeding or templating manner. In this work we seek an understanding of how particular amino acids contribute to prion disease pathogenesis and ultimately how this information can be translated to the production of more efficient therapies.

We developed a high-throughput screening method to determine the amino acid specific effects of the PrP sequence contributing to the interaction with anti-prion antibodies and alternative PrP conformations. A library of mouse PrP mutants expressed on the surface of yeast cells was screened for their binding interactions with anti-prion antibodies and beta-sheet rich PrP conformations. Those substitutions in PrP that prevented these interactions were identified by single molecule real-time (SMRT) sequencing of the screened population, providing greater than 10,000 full-length nucleotide sequences. The sequences were then aligned to the wild-type PrP gene to identify mutations. We found that optimization of the alignment scoring parameters for the Needleman-Wunsch algorithm and rejecting the lowest 10% of sequences in terms of sequence quality reduced the substitution error rate of sequencing from 7.90 x 10^-5 to 2.19 x 10^-5 and improves the statistical power of the method. By examining the entire gene sequences correlated to the protein function, we were able to obtain residue-level resolution of conformational protein-protein interaction interfaces that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity.

When the library was screened against anti-prion antibodies we found that they made contacts with discontinuous residues that are brought into close proximity when PrP adopts an alpha-helix rich and PrP^C like structure. When the library was screened against different conformations of PrP conformation specific interactions were observed. We found that antibodies ICSM18 and D18 binding was influenced by discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix was intact, while full affinity of the 6H4 antibody was dependent on the negative charge on the genetically distal but conformationally adjacent D201 residue. Furthermore, the relative enrichment of mutants correlated to the magnitude of the change in binding affinity, demonstrating how residues such as W144 were essential for binding for all three antibodies, while residues such as D201 only modestly contributed to 6H4 affinity. We observed that high affinity PrP-PrP interactions with yeast surface displayed PrP were consistently achieved when unbound PrP was folded into beta-sheet rich structures. These interactions persisted over a wide range of solution conditions and blocking conditions, and were facilitated predominantly by residues 101-111, though other regions throughout the entire protein such as residues 28-33 and 203-206 also appeared to contribute to binding. These findings reinforce the conformational importance of PrP-PrP interactions and suggest potential mechanisms by which existing and new therapeutics may act by inhibiting interactions at these sites.

In the final portion of this work we develop anti-prion antibodies for increased therapy. By yeast surface display affinity maturation, we isolated ICSM18 mutants with a greater than 300 fold increase in affinity for both recombinant PrP and for native PrP expressed by a mouse nueroblastoma cell line. When these antibodies were expressed by cells persistently infected with prions the improved affinity antibody fragments showed reduced levels of PrP in the disease conformation compared to the cells expressing the parental antibody fragment. We also developed new lead candidate antibody fragments that bind to the helix2-helix3 region that may play a role in PrP^C to PrP^Sc conversion, and are useful for structural characterization and as potential therapeutics.

Overall, a method was developed for amino-acid level characterization of protein-protein interactions and this method was applied to understand factors that contribute to PrP self-associations relevant to disease pathology and to identify the mechanism by which antibodies recognize PrP relevant to disease treatment.

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Huang, Wei-Cheng. "Protein engineering of P450BM3 by rational redesign." Thesis, University of Leicester, 2008. http://hdl.handle.net/2381/8457.

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The potential of flavocytochrome P450BM3 (CYP102A1) from Bacillus megaterium for industrial chemical transformation and biotechnological application is widely acknowledged. The crystal structures of P450BM3 with fatty acid substrates bound present non-productive modes of binding of substrate with their carbons distant from the iron and the ω-terminal end in a hydrophobic pocket at one side of the active site. Comparison between substrate-free and substrate-bound structures of P450BM3 revealed two pockets (A-arm and B-arm) in the substrate binding channel. In this thesis, A82(I/F/W) mutants in which the ‘B-arm’ pocket is filled by large hydrophobic side chains at position 82 were constructed and characterised. The A82F and A82W mutants have greater affinities for substrates (~ 800-fold) as well as being more effective catalysts of indole hydroxylation than the wild-type enzyme. The crystal structure of the haem domain of the A82F mutant with bound palmitate showed different substrate binding position, in which the substrate is closer to the haem iron than wild-type enzyme. On this basis, a second series of mutants with substitutions at position 438 as well as 82, in which the ‘A-arm’ pocket is modulated by large hydrophobic side chains, were constructed and characterised. The hydroxylation of 11-methyllaurate by wild-type was found to yield traces of the ω-hydroxylated product, which is the first observation of ω-hydroxylase activity of wild-type P450BM3 to date. The mutants with both ‘B-arm’ pocket and ‘A-arm’ pocket filled with larger hydrophobic residues (A82F-T438(V/I/L/F) mutants) demonstrated 2- to 3-fold increases in the formation of ω-hydroxyl-11-methyllaurate. Notably, the A82F-T438L and A82F-T438F mutants also presented a marked enhancement of stereo-selectivity for styrene epoxidation to generate R-styrene oxide (~ 30-fold), suggesting that not only that these mutants of P450BM3 will be valuable catalysts for synthetically useful hydroxylation reactions but also that structure-based rational redesign will be one of the most efficient tools to generate novel biocatalysts.

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Deacon, Sarah Elizabeth. "Protein expression and engineering of galactose oxidase." Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494592.

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Kennedy, Ann. "Protein engineering of an industrially-used lipase." Thesis, University of Nottingham, 2001. http://eprints.nottingham.ac.uk/11098/.

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Lipase 3 is a fungal lipase produced industrially for use as a dough-conditioning enzyme in bread making. Mutants of Lipase 3 were designed to improve enzyme specific activity and to prevent N-linked glycosylation, which was found to cause a drop in activity on industrial-scale production. These were based on three-dimensional model structures of Lipase 3 in `open' and `closed' conformational states derived from crystallographic data of fungal lipases sharing high sequence homology. Lipase variants were expressed and secreted by Pichiapastoris yeast and purified by anionexchange chromatography, which allowed the separation of two active isoforms. Analysis of the `glycosylation' mutants by SDS-PAGE and MALDI-TOF mass spectrometry implied that mutation of N-linked glycosylation sites prevented attachment of oligosaccharide groups to these sites. Mutants were characterised by measurement of specific activities with soluble and emulsified substrates and determination of kinetic constants with emulsified substrate. None of the `activity' mutants showed improved activity over wild type, and the significant drop in activity with emulsified substrate on mutation of a `lid' tryptophan residue indicated that this residue was required for interaction with long-chain triacylglycerol substrates. Specific activities and kinetic constants measured for the ‘glycosylation' mutants did not differ significantly from those of the wild type enzyme. Sigmoidal kinetic curves were observed for lipases expressed in Pichia pastoris and Aspergillus niger with emulsified substrate. Co-operativity was measured using the Hill plot and found to be positive. The possibility of a kinetic mechanism, rather than an allosteric mechanism (involving interactions between ligand binding sites), for cooperativity is discussed.

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Campbell, A. F. "Protein engineering of chymotrypsin inhibitor II (C12)." Thesis, Imperial College London, 1988. http://hdl.handle.net/10044/1/46983.

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Karydis, Thrasyvoulos. "Learning hierarchical motif embeddings for protein engineering." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/109659.

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Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 75-79).
This thesis lays the foundation for an integrated machine learning framework for the evolutionary analysis, search and design of proteins, based on a hierarchical decomposition of proteins into a set of functional motif embeddings. We introduce, CoMET - Convolutional Motif Embeddings Tool, a machine learning framework that allows the automated extraction of nonlinear motif representations from large sets of protein sequences. At the core of CoMET, lies a Deep Convolutional Neural Network, trained to learn a basis set of motif embeddings by minimizing any desired objective function. CoMET is successfully trained to extract all known motifs across Transcription Factors and CRISPR Associated proteins, without requiring any prior knowledge about the nature of the motifs or their distribution. We demonstrate that motif embeddings can model efficiently inter- and intra- family relationships. Furthermore, we provide novel protein meta-family clusters, formed by taking into account a hierarchical conserved motif phylogeny for each protein instead of a single ultra-conserved region. Lastly, we investigate the generative ability of CoMET and develop computational methods that allow the directed evolution of proteins towards altered or novel functions. We trained a highly accurate predictive model on the DNA recognition code of the Type II restriction enzymes. Based on the promising prediction results, we used the trained models to generate de novo restriction enzymes and paved the way towards the computational design of a restriction enzyme that will cut a given arbitrary DNA sequence with high precision.
by Thrasyvoulos Karydis.
S.M.

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Sutton, Samantha C. (Samantha Carol). "Engineering phosphorylation-dependent post-translational protein devices." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45205.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.
Includes bibliographical references (p. 117-127).
One goal underlying synthetic biology is to develop standard biological parts that can be reliably assembled into devices encoding higher-order functions. Here, I developed a framework for engineering post-translational devices, which are devices whose inner workings are modulated by non-covalent protein interactions and covalent protein modifications. To test the framework, I designed a scaffold for engineering post-translational devices in yeast, the Phospholocator, that can be used to assemble peptide parts in order to produce devices that couple upstream kinase activity to regulated nuclear translocation. I used the Phospholocator to design, build, and characterize a Phospholocator device, the Cdc28-Phospholocator, whose location is regulated by the activity of cyclin-dependent kinase Cdc28. I next engineered and tested a Fus3-Phospholocator device, whose location is regulated by the activity of the mitogen-activated protein kinase Fus3, in order to demonstrate that the Phospholocator scaffold supports the engineering of many post-translational devices. I used the Cdc28-Phospholocator to follow Cdc28 activity levels throughout the yeast cell cycle, thereby illustrating the utility of the Cdc28-Phospholocator as a tool for biological inquiry. To implement more complex functions, device engineers will want to connect post-translational devices to build multi-component systems. I thus developed a model for device composition that features a universal signal carrier that is both input into and output from post-translational devices. The universal signal could enable engineers to easily combine devices in any desired order, and thus build many new post-translational systems.
(cont.) I next developed a set of specifications and guidelines for designing prototypical protein parts for engineering post-translational devices that communicate via the universal signal carrier. I used the universal signal model and the corresponding set of device specifications to design and model a proof-of-principle. multi-device post-translational system, a post-translational latch, that functions as designed. Taken together, my initial experiences in engineering post-translational devices, defining universal device signals that enable device interconnectivity, and designing, modeling, and analyzing the model of a functional multi-device system, along with the work of many other groups, are sufficiently encouraging to motivate continued work on post-translational devices.
by Samantha C. Sutton.
Ph.D.

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Enever, Carolyn Louise. "Protein L : a tool for antibody engineering." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619838.

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Bocci, Marco. "Protein engineering of HGF/SF for therapy." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616080.

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Thurston, Victoria Louise. "Biophysical, structural and protein engineering studies on rabbit ileal lipid binding protein." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517865.

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Callahan, Nicholas. "Bioinformatics-Driven Enzyme Engineering: Work On Adenylate Kinase." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1420802270.

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Park, Changmoon Goddard William A. "Protein design and simulation Part I. Protein design. Part II. Protein simulation /." Diss., Pasadena, Calif. : California Institute of Technology, 1993. http://resolver.caltech.edu/CaltechTHESIS:11112009-114142428.

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Thesis (Ph. D.)--California Institute of Technology, 1993. UM #93-25,374.
Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.

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48

Halliwell, Catherine Mary. "Genetic engineering of metalloproteins." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244551.

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Kanje, Sara. "Engineering of small IgG binding domains for antibody labelling and purification." Doctoral thesis, KTH, Proteinteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-191303.

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In protein engineering, rational design and selection from combinatorial libraries are methods used to develop proteins with new or improved features. A very important protein for the biological sciences is the antibody that is used as a detecting agent in numerous laboratory assays. Antibodies used for these purposes are often ”man-made”, by immunising animals with the desired target, or by selections from combinatorial libraries. Naturally, antibodies are part of the immune defence protecting us from foreign attacks from e.g. bacteria or viruses. Some bacteria have evolved surface proteins that can bind to proteins abundant in the blood, like antibodies and serum albumin. By doing so, the bacteria can cover themselves in the host’s own proteins and through that evade being detected by the immune system. Two such proteins are Protein A from Staphylococcus aureus and Protein G from group C and G Streptococci. Both these proteins contain domains that bind to antibodies, one of which is denoted C2 (from Protein G) and another B (from Protein A). The B domain have been further engineered to the Z domain. In this thesis protein engineering has been used to develop variants of the C2 and Z domains for site-specific labelling of antibodies and for antibody purification with mild elution. By taking advantage of the domains’ inherent affinity for antibodies, engineering and design of certain amino acids or protein motifs of the domains have resulted in proteins with new properties. A photo crosslinking amino acid, p-benzoylphenylalanine, have been introduced at different positions to the C2 domain, rendering three new protein domains that can be used for site-specific labelling of antibodies at the Fc or Fab fragment. These domains were used for labelling antibodies with lanthanides and used for detection in a multiplex immunoassay. Moreover, a library of calcium-binding loops was grafted onto the Z domain and used for selection of a domain that binds antibodies in a calcium dependent manner. This engineered protein domain can be used for the purification of antibodies using milder elution conditions, by calcium removal, as compared to traditional antibody purification.

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Brown, Nicola Louise. "Engineering of an IgG-binding protein based upon protein A from Staphylococcus aureus." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295682.

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Dissertations / Theses on the topic 'Protein engineering'

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