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Engineering novel chemosensory proteins to respond to antiviral drugsTague, Elliot Parker 15 May 2021 (has links)
Cellular activities constantly change to precisely respond to their biological needs. In many cases, proteins carry out these activities because they can exhibit graded and dynamic responses to perform an array of cellular functions. To study these biological activities and to repurpose proteins for novel uses such as cell therapies, we must be able to control protein activity with synthetic inducers, such as chemical ligands. Multiple chemical inducers have been employed to achieve protein control, but there remains a need for inducer ligands that minimally interact with endogenous pathways, display high bioavailability, and are absent or minimally present from dietary sources.
In this work, we control protein activities with the Hepatitis C virus cis-protease NS3 and its numerous clinically validated, highly specific inhibitors. First, we use NS3 to create a Ligand Inducible Connection (LInC) to chemically control gene expression, protein localization and cell signaling in mammalian cells. We then extend the use of catalytically inactive NS3 as a high affinity binder in conjunction with genetically encoded approaches to inhibit NS3, including peptides and RNA aptamers. Using catalytically inactive NS3, genetically encoded peptides, and small molecule drugs, we conditionally control peptide docking with antiviral drugs. We apply this concept to control mammalian gene expression, cell signaling, enzyme activity, and develop a new mechanism for allosteric regulation of Cre recombinase. Altogether, we have developed a new toolkit for controlling diverse protein activities with highly orthogonal, antiviral drugs. / 2023-05-15T00:00:00Z
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Characterization of the CAN1 gene and its product in S. cerevisiaeAhmad, Margaret January 1987 (has links)
No description available.
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Analyzing the Sequence-Stability Landscape of the Four-helix Bundle Protein Rop: Developing High-Throughput Approaches for Combinatorial Biophysics and Protein EngineeringLavinder, Jason James 10 September 2009 (has links)
No description available.
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Protein Engineering for Biomedical MaterialsParker, Rachael N. 17 April 2017 (has links)
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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The role of proline residue to the thermostability of proteins.January 2005 (has links)
Ma Hoi-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / 摘要 --- p.III / Content --- p.IV / Abbreviations --- p.X / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Interactions that stabilize proteins --- p.1 / Chapter 1.2 --- Some common strategies of protein engineering to improve thermostability --- p.6 / Chapter 1.3 --- Ribosomal protein T. celer L30e as a study model for thermostability --- p.7 / Chapter 1.4 --- Extra proline residue is one of the insights by comparing the two proteins --- p.10 / Chapter Chapter Two --- Materials and Methods --- p.13 / Chapter 2.1 --- General Techniques --- p.13 / Chapter 2.1.1 --- Preparation of Escherichia coli competent cells --- p.13 / Chapter 2.1.2 --- Transformation of Escherichia coli competent cells --- p.14 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.14 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.14 / Chapter 2.1.5 --- DNA extraction from agarose gel electrophoresis using Viogene Gene Clean kit --- p.15 / Chapter 2.1.6 --- Plasmid DNA minipreperation by Wizard® Plus SV Minipreps DNA Purification System from Promega --- p.16 / Chapter 2.1.7 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.1.8 --- Ligation of DNA fragments --- p.18 / Chapter 2.1.9 --- Sonication of pellet resuspension --- p.18 / Chapter 2.1.10 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.19 / Chapter 2.1.11 --- Native polyacrylamide gel electrophoresis --- p.20 / Chapter 2.1.12 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.22 / Chapter 2.1.13 --- Protein Concentration determination --- p.22 / Chapter 2.2 --- Cloning the Mutant Genes --- p.22 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.22 / Chapter 2.2.1.1 --- Generation of full length mutant gene by megaprimer --- p.23 / Chapter 2.2.1.2 --- Generation of mutant gene by QuikChange® Site-Directed Mutagenesis Kit from Stratagene --- p.26 / Chapter 2.2.2 --- Restriction Digestion of DNA --- p.27 / Chapter 2.2.3 --- Ligation of DNA fragments --- p.27 / Chapter 2.2.4 --- Screening for successful inserted plasmid clones from ligation reactions --- p.28 / Chapter 2.2.4.1 --- By PCR --- p.28 / Chapter 2.2.4.2 --- By restriction digestion --- p.28 / Chapter 2.2.5 --- DNA sequencing --- p.29 / Chapter 2.3 --- Expression and Purification of Protein --- p.29 / Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.29 / Chapter 2.3.2 --- Purification of recombinant wild type TRP and mutants --- p.30 / Chapter 2.3.3 --- Purification of recombinant wild type YRP and mutants --- p.32 / Chapter 2.4 --- Thermodynamic Studies by Circular Dichroism (CD) Spectrometry --- p.34 / Chapter 2.4.1 --- Thermodynamic studies by guanidine-induced denaturations --- p.34 / Chapter 2.4.2 --- Themodynamic studies by thermal denaturations --- p.36 / Chapter 2.4.3 --- ACp measurement of the TRP mutants --- p.37 / Chapter 2.4.3.1 --- By Gibbs-Helmholtz analysis --- p.37 / Chapter 2.4.3.2 --- By van't Hoff analysis --- p.37 / Chapter 2.5 --- Crystal Screen for the Mutant T. celer L30e --- p.38 / Chapter 2.5.1 --- T. celer L30e Pro→Ala and Pro→Gly mutants --- p.38 / Chapter 2.5.2 --- Yeast L30e K65P mutant --- p.38 / Chapter 2.6 --- Sequences of Primers --- p.39 / Chapter 2.6.1 --- Primers for TRP and its mutants --- p.39 / Chapter 2.6.2 --- Primers for YRP and its mutantsReagents and buffers --- p.40 / Chapter 2.7 --- Reagents and Buffers --- p.40 / Chapter 2.7.1 --- Reagents for competent cell preparation --- p.40 / Chapter 2.7.2 --- Nucleic acid eletrophoresis buffers --- p.41 / Chapter 2.7.3 --- Media for bacterial culture --- p.41 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.42 / Chapter 2.7.5 --- Buffers for TRP purification --- p.44 / Chapter 2.7.6 --- Buffers for YRP purification --- p.45 / Chapter 2.7.7 --- Buffer for Circular Dichroism (CD) Spectrometry --- p.46 / Chapter Chapter Three --- Results --- p.48 / Chapter 3.1 --- "Cloning, expression and purification of the mutant proteins" --- p.48 / Chapter 3.1.1 --- "Mutagenesis, cloning and purification of the thermophilic proteins - T. celer L30e protein and its mutants" --- p.48 / Chapter 3.1.2 --- "Mutagenesis, cloning and purification of the mesophilic proteins - yeast L30e protein and its mutants" --- p.52 / Chapter 3.2 --- Stability of Pro→Ala/Gly mutants of T. celer L30e at 298K --- p.55 / Chapter 3.2.1 --- Design of alanine and glycine mutants from thermophilic homologue --- p.55 / Chapter 3.2.2 --- "Among alanine mutants, only P59A was destabilized" --- p.55 / Chapter 3.2.3 --- Ala→Gly mutations destabilized the protein --- p.59 / Chapter 3.3 --- Stability of Xaa→Pro mutants of yeast L30e at 298K --- p.61 / Chapter 3.3.1 --- Design of proline mutants from mesophilic homologue --- p.61 / Chapter 3.3.2 --- "K65P, corresponding to P59 in T. celer L30e, stabilized yeast L30e" --- p.62 / Chapter 3.3.3 --- Yeast L30e mutated with thermophilic consensus sequence did not give a more stable protein --- p.65 / Chapter 3.4 --- Temperature dependency of the stability of the mutants of T. celer L30e --- p.67 / Chapter 3.4.1 --- The trend of ΔGU was consistence through 25 to 75°C --- p.67 / Chapter 3.4.2 --- Melting temperatures of T. celer mutants determined by thermal denaturations --- p.68 / Chapter 3.5 --- pH dependency of melting temperatures --- p.75 / Chapter 3.5.1 --- ΔCP values of the P59A/G mutants determined by van't HofF's analyses increased significantly --- p.77 / Chapter 3.6 --- No structural change was observed in the crystal structure of P59A --- p.80 / Chapter Chapter Four --- Discussion --- p.84 / Chapter 4.1 --- The trend of stability from guanidine-induced denaturation agreed with that from thermal denaturations --- p.86 / Chapter 4.2 --- The magnitude of destabilization of P59A and Ala→Gly mutation was consistent with the expected destabilization due to entropy --- p.87 / Chapter 4.3 --- Entropic effect had little effect for residues in flexible region --- p.93 / Chapter 4.4 --- Stabilization forces that compensate the entropic effect --- p.96 / Chapter 4.5 --- Compensatory stabilization due to the release of amide group --- p.99 / Chapter 4.5.1 --- Intra-molecular H-bond in P88A --- p.99 / Chapter 4.5.2 --- Solvent-protein H-bond in P43A --- p.103 / Chapter 4.6 --- Consensus concept was not applicable in our model --- p.110 / Chapter 4.7 --- "Pro→Ala mutation destabilized the protein increase the protein's ACP value, however enthalpy and entropy change were difficult to be decomposed" --- p.111 / Chapter 4.8 --- Concluding Remarks --- p.112 / References --- p.113
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Applications of phage-displayed antibody library for antibody discovery and engineering. / CUHK electronic theses & dissertations collectionJanuary 2008 (has links)
Antibodies are one of the most useful molecules with affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human diseases. In recent years, phage-displayed antibody library has been widely adopted to select tailor-made antibodies in a fast, high-throughput mode, as an alternative of traditional hybridoma technology. Although phage display has been introduced for about 20 years, the applications and development of this technology still have a rich space to be explored. / Attempts are made in the present study to extend three applications of the phage displayed antibody library in antibody discovery and engineering. Firstly, a CDR3-randomized phage-displayed scFv library was constructed from genomic DNA of mouse. Following biopanning, anti-peptide of mas oncoprotein scFvs were isolated and identified. These results illustrate the potential use of the genomic phage-displayed library for anti-peptide antibodies selection. Secondly, we described the isolation of anti-idiotypic scFvs against a chimeric anti-CD22 mAb from an immunized phage-displayed scFv library. The isolated anti-Id scFvs were able to capture the immune response of chimeric anti-CD22 mAb with high specificity. This reagent will enhance our understanding of the therapeutic mechanism of anti-CD22 mAb in non-Hodgkin's lymphoma treatment, and may be applied to probe the pharmacokinetics, tissue distribution, and modulation of anti-CD22 mAb in vivo. / In conclusion, we have attempted various approaches to identify specific anti-peptide scFvs, anti-idiotypic scFvs and passive anti-tumor scFvs. These results extend the applications of phage display technology in antibody discovery and engineering. / Our approach enables us to isolate selective and sensitive anti-idiotypic antibodies and could be exploited for other antibodies with clinical and biological applications. Thirdly, we profile a strategy to select and identify markers on tumor cell surface using phage-displayed antibodies from mice bearing xenograft tumor. Our data imply that passive antibodies in cancer patients may be obtained from the immune repertoire of cancer patients. Besides, we found a cell surface antigen was up-regulated more than 3-fold in mas-expressing cells. We further use the targeting antibody to construct a tumor endoprotease-activated immunotoxin. / Zhao, Qi. / Adviser: Wing-Tai Cheung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3499. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 227-250). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
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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 spektroskopiBlom, Lillemor January 2008 (has links)
<p>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.</p>
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Engineered human cytochrome c : investigation of superoxide and protein-protein interaction and application in bioelectronic systemsWegerich, Franziska January 2010 (has links)
The aim of this thesis is the design, expression and purification of human cytochrome c mutants and their characterization with regard to electrochemical and structural properties as well as with respect to the reaction with the superoxide radical and the selected proteins sulfite oxidase from human and fungi bilirubin oxidase. All three interaction partners are studied here for the first time with human cyt c and with mutant forms of cyt c.
A further aim is the incorporation of the different cyt c forms in two bioelectronic systems: an electrochemical superoxide biosensor with an enhanced sensitivity and a protein multilayer assembly with and without bilirubin oxidase on electrodes.
The first part of the thesis is dedicated to the design, expression and characterization of the mutants. A focus is here the electrochemical characterization of the protein in solution and immobilized on electrodes. Further the reaction of these mutants with superoxide was investigated and the possible reaction mechanisms are discussed.
In the second part of the work an amperometric superoxide biosensor with selected human cytochrome c mutants was constructed and the performance of the sensor electrodes was studied. The human wild-type and four of the five mutant electrodes could be applied successfully for the detection of the superoxide radical.
In the third part of the thesis the reaction of horse heart cyt c, the human wild-type and seven human cyt c mutants with the two proteins sulfite oxidase and bilirubin oxidase was studied electrochemically and the influence of the mutations on the electron transfer reactions was discussed.
Finally protein multilayer electrodes with different cyt form including the mutant forms G77K and N70K which exhibit different reaction rates towards BOD were investigated and BOD together with the wild-type and engineered cyt c was embedded in the multilayer assembly. The relevant electron transfer steps and the kinetic behavior of the multilayer electrodes are investigated since the functionality of electroactive multilayer assemblies with incorporated redox proteins is often limited by the electron transfer abilities of the proteins within the multilayer. The formation via the layer-by-layer technique and the kinetic behavior of the mono and bi-protein multilayer system are studied by SPR and cyclic voltammetry.
In conclusion this thesis shows that protein engineering is a helpful instrument to study protein reactions as well as electron transfer mechanisms of complex bioelectronic systems (such as bi-protein multilayers). Furthermore, the possibility to design tailored recognition elements for the construction of biosensors with an improved performance is demonstrated. / Ziel dieser Arbeit ist es genetisch veränderte Formen von humanem Cytochrom c herzustellen und diese einerseits hinsichtlich der Reaktion mit dem Sauerstoff-Radikal Superoxid aber auch mit anderen Proteinen zu untersuchen. Zusätzlich sollen die verschiedenen Protein-Mutanten in neuartige bioelektronische Systeme eingebracht werden.
Es wurden insgesamt 20 Cytochrome c Mutanten designt, rekombinant exprimiert und aufgereinigt. Es konnte in dieser Arbeit gezeigt werden, dass sich die Reaktion von Cytochrom c mit dem negativ geladenen Superoxid durch gezielte Mutationen, die zusätzliche positive Ladungen in das Molekül bringen, um bis zu 30 % erhöhen lässt. Es wurde aber auch deutlich, dass andere Eigenschaften des Proteins sowie dessen Struktur durch die Mutationen geändert werden können.
Cytochrom c Mutanten mit einer erhöhten Reaktionsrate mit Superoxid konnten erfolgreich in einen Superoxid-Biosensor mit erhöhter Sensitivität eingebracht werden.
Weiterhin wurde einige Mutanten hinsichtlich Ihrer Interaktion mit den zwei Enzymen Sulfitoxidase und Bilirubinoxidase untersucht. Hier konnten ebenfalls unterschiedliche Reaktivitäten festgestellt werden.
Schließlich wurden ausgewählte Protein-Varianten mit und ohne den zuvor untersuchten Enzymen in ein Multischicht-Elektroden-System eingebettet und dessen kinetisches Verhalten untersucht. Es wurde gefunden, dass die Schnelligkeit mit der Cytochrom c mit sich selbst Elektronen austauschen kann, eine Limitierung der Größenordnung der katalytischen Ströme darstellt. Diese Selbstaustausschrate wurde durch die eingeführten Mutationen verändert.
So verdeutlicht diese Arbeit, dass „Protein-Engineering“ ein gutes Hilfsmittel sein kann, um einerseits Proteinreaktionen und komplexe Elektronentransferreaktionen in Multischichten zu untersuchen, aber auch ein potentes Werkzeug darstellt mit dem zugeschnittene Biokomponenten für Sensoren mit erhöhter Leistungsfähigkeit generiert werden können.
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Tools for Maximizing the Efficiency of Protein EngineeringPolizzi, Karen Marie 14 November 2005 (has links)
Biocatalysts offer advantages over their chemical counterparts in terms of their high enantioselectivity and the opportunity to develop more environmentally friendly processes. However, the widespread adoption of biocatalytic processes is hampered by the long development times for enzymes with novel and sufficient activity and adequate stability under operating conditions. Protein engineering, while extremely useful for modifying the properties of protein catalysts in select cases, still cannot be performed rapidly enough for many applications. In order for biocatalysts to become a competitive alternative to chemical catalysts, new tools to make the tailoring of biocatalysts by protein engineering methods speedier and more efficient are necessary. The aim of this work was to develop methods to aid in the faster production of novel biocatalysts.
Protein engineering involves two steps: the generation of diversity and the screening or selection of variants with the desired properties. Both of these must be targeted to create a faster protein engineering process. In the case of the former, this work sought to clone and overexpress some template enzymes which would create smaller, more manageable libraries of mutants with a higher likelihood of function by the manipulation of a few focused amino acid residues. For the latter, this work developed and validated a Monte-Carlo simulation model of pooling to increase screening throughput and created a set of vectors to aid in high-throughput screening by eliminating unwanted mutants from the assay procedure entirely.
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Engineering Protein Molecular Switches To Regulate Gene Expression with Small MoleculesRohatgi, Priyanka 29 November 2006 (has links)
Small molecule dependent molecular switches that control gene expression are important tool in understanding biological cellular processes and for regulating gene therapy. Nuclear receptors are ligand activated transcription factors that have been engineered to selectively respond to synthetic ligands and used as regulators of gene expression. In this work the retinoid X receptor (RXR), has been used to develop an inducible molecular switch with a near drug like compound LG335. Three RXR variants (Q275C; I310M; F313I), (I268A; I310A; F313A; L436F), (I268V; A272V; I310M; F313S; L436M) were created via site-directed mutagenesis and a structure based approach, such that they preferentially bind to the synthetic ligand LG335 and not its natural ligand, 9-cis retinoic acid. These variants show reverse ligand specificity as designed and have an EC50 for LG335 of 80 nM, 30 nM, 180 nM, respectively. The ligand binding domains of the RXR variants were fused to a yeast transcription factor Gal4 DNA binding domain. This modified chimeric fusion protein showed reverse response element specificity as designed and recognized the Gal4 response element instead of the RXR response element. The modified RXR protein did not heterodimerize with wild type RXR or with other nuclear receptor such as retinoic acid receptor. These RXR-based molecular switches were tested in retroviral vectors using firefly luciferase and green fluorescence protein and they maintain their inducible behavior with LG335. These experiments demonstrate the orthogonality of RXR variants and their possible use in regulating gene therapy.
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