Browsing by Subject "Protein engineering"
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Item Advancing high-throughput antibody discovery and engineering(2014-05) Kluwe, Christien Alexandre; Ellington, Andrew D.; Georgiou, GeorgeThe development of hybridoma technology nearly forty years ago set the foundation for the use of antibodies in the life sciences. Subsequent advances in recombinant DNA technology have allowed us to adapt antibody genes to various screening systems, greatly increasing the throughput and specialized applications for which these complex biomolecules can be adapted. While selection systems are a powerful tool for discovery and evolution, they can be slow and prone to unintended biases. We see computational approaches as an efficient process for rapid discovery and engineering of antibodies. This is particularly relevant for biodefense and emerging infectious disease applications, for which time is a valuable commodity. In the first chapter of this work, we examine computational protocols for ‘supercharging’ proteins. This process resurfaces the target protein, adding charged moieties to impart specialized functions such as thermoresistance and cell penetration. Current algorithms for resurfacing proteins are static, treating each mutation as an event within a vacuum. The net result is that while several variants can be created, each must be tested experimentally to ensure the resultant protein is functional. In many cases, the designed proteins were severely impaired or incapable of folding. We hypothesize that a more dynamic approach, keeping an eye on energetics and the consequences of mutations will yield a more efficient and robust method for supercharging, successfully adding charges to proteins while minimizing deleterious effects. We continue on this theme applying the successful algorithm to supercharging antibodies for increased function. Utilizing the MS2 model biosensor system, we rationally engineer charges onto the surface of an antibody fragment, increasing thermoresistance, minimizing destabilizing effects, and in some cases actually increasing affinity. Finally, we apply next-generation sequencing approaches to the rapid discovery of antibodies directed against the Zaire Ebolavirus species. We utilize a local immunization strategy to generate a polarized antibody repertoire that is then sequenced to provide a database of antigen-specific variants. This repertoire is probed in silico and individual antibodies selected for analysis, bypassing time- and resource-consuming selection experiments.Item Delivery of vaccines and therapeutics to treat infectious diseases(2012-12) Khan, Tarik Ali; Maynard, Jennifer Anne, 1974-Efficient delivery of vaccines and therapeutic agents in vivo is a critical aspect for ensuring a desired immunological or biological response is achieved. This work focuses on developing effective delivery systems for vaccines and therapeutics to achieve biological potency while maintaining patient friendly administration. Traditional vaccines are administered via parenteral injection, which requires skilled personnel for administration and does not elicit strong mucosal immune responses. An alternative approach is to develop an oral vaccine; however, this requires antigens to be protected during transit through the gastrointestinal tract and be transported across specialized intestinal sampling cells called M cells. These M cells are extremely rare, making them an important target for oral vaccines. The protein invasin, from Yersinia psuedotuberculosis, naturally binds [mathematical symbols] integrin, a receptor found exclusively on M cells within the gastrointestinal tract. Therefore, we generated combinatorial libraries of invasin, followed by a directed evolution and high-throughput screening strategy to identify invasin variants with increased affinity towards [mathematical symbols] integrin. This process led to the creation of an invasin variant exhibiting a nine-fold decrease in EC₅₀, which could be used for targeted oral vaccine systems. In order to test for increased vaccine efficacy due to the engineered invasin ligand, we developed a polymeric microparticle delivery system. These microparticles were formulated to encapsulate the model antigen ovalbumin and be decorated with the invasin targeting ligands. To measure physiological trafficking and intestinal retention, novel fluorescent nanocrystals were loaded into particles conjugated to invasin. These nanocrystals served as a contrast agent for in vivo imaging in mice. While these particles were unsuccessful in generating an antibody response toward ovalbumin when administered to mice, a response directed to the targeting ligand itself was observed. These findings provide insights for further optimizing a delivery system for oral vaccination. In addition to developing an oral vaccine delivery system, we created a high concentration therapeutic protein formulation, suitable for low-volume subcutaneous administration. By adding crowding agents, we were able to generate reversible protein nanoclusters with low viscosity. These nanoclusters were found to revert to monomer upon dilution and pharmacokinetic profiles similar to solutions.Item Engineering a novel human methionine degrading enzyme as a broadly effective cancer therapeutic(2014-08) Paley, Olga M.; Georgiou, George; Iverson, Brent; Alper, Hal S; Maynard, Jennifer; Johnson, Kenneth AMany cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic.Item Engineering a protein for peptide detection and allosteric activation(2010-05) Lewis, Marsha Jane, 1970-; Iverson, Brent L.; Georgiou, George; Fast, Walter; Maynard, Jennifer; Ren, PengyuStrategies for the engineering of allosteric proteins, which are proteins that bind ligands at a specific site other than the reaction site and affect the reaction activity, are still being perfected. There have been allosteric proteins successfully engineered based on the hypothesis that the two allosterically related sites are distinct, modular domains and use trial and error to construct and test novel protein domain fusions for allostery. This work uses laboratory evolution to engineer the peptide binding affinity of the protein binding domain of the allosteric E. coli protease DegS. The protein binding domain is a PDZ domain (named for Postsynaptic density protein (PSD-95), Discs-large protein (Dlg), and Zonula occludens-1 (ZO-1)) that binds the C-terminus of unfolded outer membrane porins. Combinatorial libraries of PDZ domain variants were displayed anchored to the periplasmic membrane of E. coli. The cells were permeabilized and incubated with fluorescent peptide ligands. PDZ domains were screened by flow cytometry for binding to the target peptide ligands. The PDZ domain binding affinity was improved by 20-fold for the peptide ligand that represents the physiological ligand; and the PDZ domain binding affinity was expanded to accommodate a negatively charged residue in a novel peptide ligand. The E. coli anchored peripalsmic expression (APEx) methodology in conjunction with flow cytometry had not previously been used to modify the binding affinity of a PDZ domain. The selected PDZ domain variants were then fused to the wild-type DegS protease domain and analyzed to determine if allosteric activation was made more sensitive to the native ligand or altered to respond to the novel peptide ligand. Interestingly, the DegS fusion protein with the PDZ variant containing the most subtle mutations retained a degree of allostery for the physiological peptide ligand and obtained a degree of allostery for the novel activating peptide ligand. Other selected PDZ variants with additional and expected mutations in the ligand binding site did not respond allosterically to the peptide ligands and the respective DegS fusions were constitutively active, suggesting that the amino acid network linking the allosteric binding event to protease activity is intricately integrated.Item Engineering antibody and T cell receptor fragments : from specificity design to optimization of stability and affinity(2014-12) Entzminger, Kevin Clifford; Maynard, Jennifer Anne, 1974-B and T cells comprise the two major arms of the adaptive immune response tasked with clearing and preventing infection; molecular recognition in these cells occurs through antibodies and T cell receptors (TCRs), respectively. Highly successful therapeutics, clinical diagnostics and laboratory tools have been engineered from fragments of these parent molecules. The binding specificity, affinity and biophysical characteristics of these fragments determine their potential applications and resulting efficacies. Thus engineering desired properties into antibody and TCR fragments is a major concern of the multi-billion dollar biopharmaceutical industry. Toward this goal, we (1) designed antibody specificity using a novel computational method, (2) engineered thermoresistant Fabs by phage-based selection and (3) modulated binding kinetics for a single-chain TCR. In the first study, de novo modeling was used to generate libraries of FLAG peptide-binding single-chain antibodies. Phage-based screening identified a dominant design, and activity was confirmed after conversion to soluble Fab format. Bioinformatics analysis revealed potential areas for design process improvement. We present the first experimental validation of this in silico design method, which can be used to guide future antibody specificity engineering efforts. In the second study, the variable heavy chain of a moderately stable EE peptide-binding Fab was subjected to random mutagenesis, and variants were selected for resistance to heat inactivation. Thermoresistant clones where biophysically characterized, and structural analysis of selected mutations suggested general mechanisms of stabilization. Framework mutations conferring thermoresistance can be grafted to other antibodies in future Fab stabilization work. In the third study, TCR fragment binding kinetics for a clonotypic antibody were modulated by varying valence during phage-based selection. Binding affinity and kinetics for representative variants depended on the display format used during selection, and all TCR fragments retained binding to native pMHC antigen. This work demonstrates a general engineering platform for tuning protein-protein interactions. Using a combination of computational design and phage-based screening, we have identified antibodies and TCR fragments with improved binding properties or biophysical characteristics. The optimized variants possess a wider range of potential applications compared to their parent molecules, and we detail engineering methods likely to be useful in the engineering of many other protein-based therapeutics.Item The engineering of de novo pathways for oxidative protein folding in Escherichia coli(2006) Masip, Lluis; Georgiou, GeorgeItem Engineering peptide specific hyper-crystallizable antibody fragments (scFv) as potential chaperones for co-crystallization(2010-12) Pai, Jennifer Chentzu; Maynard, Jennifer Anne, 1974-; Alper, Hal; Georgiou, George; Truskett, Thomas M.; Robertus, Jon D.Hydrophobic membrane proteins perform a variety of important functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. We aim to develop a toolbox of peptide specific single-chain antibody fragment chaperones engineered for hyper-crystallizability. These peptide sequences can be introduced into various regions of membrane proteins without interfering with protein function. The resulting protein-chaperone complex is expected to form a crystal lattice mediated by chaperone interactions. We have developed candidate scFv chaperone proteins binding hexa-histidine (His6) and EYMPME (EE) tags with improved biophysical features influencing crystallization propensity, including peptide affinity, stability and solubility. The scFv libraries were generated using a novel ligation-free technique, MegAnneal, allowing us to rapidly generate large libraries based on 3D5 scFv. We identified two candidate chaperones, 3D5/His_683, specific for His6 and 3D5/EE_48, specific for EE tags. Variants exhibit high solubility (up to 16.6 mg/ml) and nanomolar peptide affinities; complexes of 3D5/EE_48 with EE-tagged proteins were isolated by gel filtration. We have developed design rules for EE peptide placement at terminal, inter-domain or internal loop regions of the target protein to balance peptide accessibility for chaperone binding while retaining rigid protein-chaperone complexes suitable for crystallization. The 3D5/ His_683 crystallized in four different conditions, utilizing multiple space groups. The 3D5/EE_48 scFv was crystallized (3.1 Å), revealing a ~52 Å channel in the crystal lattice, which may accommodate a small peptide-tagged target protein. Our evolution experiments altered scFv surface residues, resulting in use of different crystallization contacts. Analysis of these crystal contacts and those used by crystallized 14B7 scFv variants, led us to postulate that lattice formation is driven by strong crystal contacts. To test this hypothesis, we introduced amino acid changes expected to reduce the affinity of the 3D5/EE_48 energetically dominant crystal contacts. This approach to crystal contact engineering may allow semi-rational control over lattice networks preferred by scFv chaperones. Co-crystallization trials with model proteins are on-going. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.Item The evolution and engineering of T7 RNA polymerase(2014-08) Meyer, Adam Joshua; Ellington, Andrew D.; Alper, Hal; Barrick, Jeffrey; Bull, James; Fast, WalterT7 RNA polymerase is a single protein capable of driving transcription from a simple promoter in virtually any context. This has made it a powerful tool in a range of biotechnology applications. In this work, previous efforts to evolve or engineer T7 RNA polymerase are reviewed. This work is then expanded upon, first with the development of a method for the cell-free evolution of T7 RNA polymerase based on the functioning of an autogene. The autogene is a transcriptional feedback circuit in which active T7 RNA polymerase proteins transcribe their own gene, resulting in exponential amplification of their genetic information. While this system is doomed by an error catastrophe, this can be delayed by the use of in vitro compartmentalization. In response to the limits of the autogene, a novel directed evolution approach termed compartmentalized partnered replication (CPR) is presented. CPR couples the in vivo functionality of a gene to its subsequent in vitro amplification by emulsion PCR. The use of CPR to generate a panel of six versions of T7 RNA polymerase, each specific to one of six promoters, is described. Separately, a rational engineering approach, taken to facilitate the high-yield transcription of fully 2′-modified RNA, is detailed. Two sets of mutations to T7 RNA polymerase, previously known to confer thermal stability and enhance promoter clearance respectively, can be used to enhance the activity of existing T7 RNA polymerase mutants that utilize non-standard nucleotides as their substrates. Next, CPR and random mutagenesis is used to populate the functional fitness landscape of T7 RNA polymerase. This neutral drift library is then challenged to increase the processivity of T7 RNA polymerase, enabling long-range transcription. Finally, the lessons that can be learned about T7 RNA polymerase specifically and molecular evolution and protein engineering generally are discussed.Item Evolved enzymes for cancer therapeutics and orthogonal systems(2013-08) Lu, Wei-Cheng; Ellington, Andrew D.Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP.Item Expanding the genetic code in mammalian cells(2011-08) Xiang, Liang; Zhang, Zhiwen Jonathan; Georgiou, George; Roy, Krishnendu; Ren, Pengyu; Yin, WhitneyProteins are diverse polymers of covalently linked amino acids. They play a role in almost every biological process that occurs within an organism. Twenty different amino acids are genetically encoded by mammalian cells to build proteins. The sequence of these amino acids determines the protein’s final shape, structure, and function. Modern molecular cloning techniques allow for the genetic encoding and expression of mutant proteins that have one or more amino acids replaced with one of the others. The roles of individual amino acids in a protein can therefore be studied. Proteins with novel functions have also been designed or evolved using this technology. However, the genetic code is limited to the twenty natural amino acids. Nonnatural amino acids have unique side groups that not found on any of the twenty natural amino acids. They can be site-specifically incorporated using a mutant orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. Each pair only allows for one type of nonnatural amino acid to be genetically encoded. This technology has resulted in the incorporation of over fifty different types of nonnatural amino acids into proteins in prokaryotic and eukaryotic cells. Unfortunately, most of these pairs are not orthogonal outside of prokaryotic systems and only a few have been developed for mammalian cells. To create more mammalian pairs a nonnatural aaRS has to be evolved and screened in a cumbersome process. In this dissertation an approach is outlined that can be used to change the orthogonality of existing nonnatural suppressor tRNA/aaRS pairs. As a result of the orthogonality change many previously unavailable pairs can be shuttled into mammalian cells. The ability to genetically encode a 21st amino acid is a powerful tool in the study and engineering of proteins.Item FACS: a high throughput method for protein export and engineering(2006) Ribnicky, Brian Michael; Georgiou, GeorgeItem Modulators of protein kinase C activity alter 5-hydroxytryptamine-3 receptor function(Texas Tech University, 1999-05) Coultrap, Steven JesseThe 5-hydroxytryptamine3 (5-HT3) receptor is a member of the superfamily of ligand-gated ion channels. Other members of this family include the GABAA, glycine, and nicotinic acetylcholine receptors. The function of many members of this family is regulated by phosphorylation of the receptor by intracellular protein kinases. Protein kinase C (PKC) is a well-studied protein kinase, that phosphorylates proteins on serine or threonine residues and is stimulated by activation of many plasma membrane bound receptors. PKC phosphorylates and modulates the function of many ligand-gated ion channels. Several compounds exist that activate or inhibit PKC activity independent of receptor activation. The present study explores the modulation of the 5-HT3 receptor by several of these compounds. Phorbol 12-myristate, 13-acetate (PMA) stimulates PKC activity and enhances 5-HT3 receptor function. Bisindolylmaleimide I, is a selective PKC inhibitor that decreases 5-HT3 receptor function. The goal of this study is to determine if the increase in 5-HT3 receptor mediated current following treatment with PMA is due to phosphorylation of the receptor by PKC and to determine if the inhibition of 5-HT3 receptor mediated current seen with bisindolylmaleimide I is due to inhibition of PKC activity. In order to answer these questions, recombinant murine 5-HT3 receptors were expressed in Xenopus laevis oocytes and studied by two-electrode voltage-clamp recordings. The enhancement in 5-HT3 receptor function by PMA follows a time and concentration dependency consistent with the involvement of PKC. Furthermore, the PKC inhibitor PKCI partially inhibits the PMA induced potentiation of current. Thus, the PMA effect is at least partially dependent on PKC activity. A 5-HT3 receptor was created in which all of the intracellular serine and threonine residues were mutated to non-phosphorlatable alanine residues (the XIST mutant receptor. The PMA induced increase in receptor function of the XIST mutant did not differ from the wild-type receptor. Howe\er. mutation of a t>Tosine residue, to a non-phosphorlatable phenylalanine residue, on the NTST mutant resulted in a reduction in the PMA effect. Paradoxically, mutation of the tyrosine residue alone had no effect on PMA dependent potentiation of current. Treatment with lavendustin A. a tyrosine kinase inhibitor, reduced the PMA enhancement of the MSI mutant, but not the wild-type receptor. Thus, PMA treatment leads to activation of PKC and a tyrosine kinase. Phosphorylation of the 5-HT3 receptor by a tyrosine kinase is likely, partially responsible for the increase in current. Some serine and/or threonine residues on the 5-HT3 receptor are also involved in the increase in receptor function, but it is not clear if they are substrates for PKC-dependent phosphorylation or part of a binding site for an associated phosphoprotein. The PKC inhibitor, bisindolylmaleimide I. decreases 5-HT3 receptor function. However, the PKC inhibitors, chelerythrine and calphostin C, did not alter 5-HT3 receptor function. It is shown that the inhibition by bisindolylmaleimide I is due to competition with 5-HT at the agonist binding site rather than through a PKC dependent mechanism. Bisindohlmaleimide I decreased the potency, without altering the efficacy, of 5-HT3. A Schild plot was generated and the slope determined to be -1. Bisindolylmaleimide I also displaced binding of the selective 5-HT; receptor antagonist. [3^H]GR65630. All of these observations are consistent with bisindolylmaleimide I acting as a competitive entagonist at the 5-HT3 receptor.Item Next generation approaches toward engineering therapeutic proteases(2012-05) Pogson, Mark Wilson; Iverson, Brent L.; Georgiou, GeorgeEngineering protease substrate specificity and selectivity has the potential to yield entirely new possibilities in the analytical, biotechnological, and therapeutic domains. For example, therapeutic applications can be envisioned in which engineered proteases could replace antibodies by irreversibly inactivating a large excess of disease-associated target proteins in a catalytic fashion. Technological advances in molecular biology have made laboratory-based evolution techniques for protein engineering readily accessible. However, the ability to interrogate the activities and substrate preference of large numbers of protease variants is predicated on the availability of quantitative high-throughput assays that maintain the essential link between genotype and phenotype. In this work we have investigated a variety of novel single cell fluorescence assays and selections for engineering protease substrate specificity and selectivity, and demonstrated the utility of some of these systems for the engineering of novel enzymes. The second chapter of this dissertation reports the isolation of a highly active ([chemical formula]) variant of the Escherichia coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine, sulfotyrosine, phosphotyrosine and phosphoserine. The isolation of protease variants that can discriminate between substrates based on the posttranslational modification of Tyr was made possible by implementing a multi-color flow cytometric assay using multiple simultaneous counter-selection substrates for the screening of large mutant libraries. While primary sequence recognition may suffice for proteomic applications, many therapeutic applications of engineered proteases will require the cleavage of folded protein targets. Unfortunately, we have found that engineered proteases that can cleave peptides very efficiently are often unable to digest the same sequences inserted into the loop regions of a folded protein. The logical conclusion, then, is that an entire target protein or at least a protein domain, rather than peptide segments, must be incorporated into protease engineering screening assays. As a critical first step toward the development of next generation, single cell screening systems for therapeutic protease engineering we have developed novel assays that exploit cell surface capture of exogenous protein substrates. One assay (Chapter 3) relies on an autoinhibited protein fusion that capitalizes on the p53 antagonist MDM2 as a detector of protease activity in addition to its utility as a counter-selection substrate. Using this system we successfully isolated OmpT variants that selectively cleave a designed site within our autoinhibited substrate. A second high-throughput screen (Chapter 4) monitors native protein cleavage. Target proteins are captured at the cell surface using a polycationic tail, incorporating counter-selection, and the proteolytic state of the substrate can be monitored using epitope tags fused to the N-and C-termini and fluorescently labeled anti-epitope tag antibodies.Item Novel high-throughput screening methods for the engineering of hydrolases(2011-05) Gebhard, Mark Christopher; Georgiou, George; Alper, Hal; Ellington, Andrew D.; Iverson, Brent L.; Maynard, Jennifer A.Enzyme engineering relies on changes in the amino acid sequence of an enzyme to give rise to improvements in catalytic activity, substrate specificity, thermostability, and enantioselectivity. However, beneficial amino acid substitutions in proteins are difficult to rationally predict. Large numbers of enzyme variants containing random amino acid substitutions are screened in a high throughput manner to isolate improved enzymes. Identifying improved enzymes from the resulting library of randomized variants is a current challenge in protein engineering. This work focuses on the development of high-throughput screens for a class of enzymes called hydrolases, and in particular, proteases and esterases. In the first part of this work, we have developed an assay for detecting protease activity in the cytoplasm of Escherichia coli by exploiting the SsrA protein degradation pathway and flow cytometry. In this method, a protease-cleavable linker is inserted between a fusion protein consisting of GFP and the SsrA degradation tag. The SsrA-tagged fusion protein is degraded in the cell unless a co-expressed protease cleaves the linker conferring higher cellular fluorescence. The assay can detect specific cleavage of substrates by TEV protease and human caspase-8. To apply the screen for protease engineering, we sought to evolve a TEV protease variant that has altered P1 specificity. However, in screening enzyme libraries, the clones we recovered were found to be false positives in that they did not express protease variants with the requisite specificities. These experiments provided valuable information on physiological and chemical parameters that can be employed to optimize the screen for directed evolution of novel protease activities. In the second part of this work, single bacterial cells, expressing an esterase in the periplasm, were compartmentalized in aqueous droplets of a water-in-oil emulsion also containing a fluorogenic ester substrate. The primary water-in-oil emulsion was then re-emulsified to form a water-in-oil-in-water double emulsion which was capable of being analyzed and sorted by flow cytometry. This method was used to enrich cells expressing an esterase with activity towards fluorescein dibutyrate from an excess of cells expressing an esterase with no activity. A 50-fold enrichment was achieved in one round of sorting, demonstrating the potential of this method for use as a high-throughput screen for esterase activity. This method is suitable for engineering esterases with novel catalytic specificities or higher stabilitItem Oral delivery of protein-transporter bioconjugates using intelligent complexation hydrogels(2008-12) Shofner, Justin Patrick, 1983-; Peppas, Nicholas A., 1948-; Brodbelt, Jennifer S.Several polymer systems including P(MAA-g-EG) and P(MAA-co-NVP) with crosslinking agents TEGDMA and PEGDMA1000, monomer-to-solvent ratios of 67:33, 60:40, and 50:50, and particle sizes of <75 microns, 90-150 microns, and 150-212 microns were synthesized for use with protein-transporter conjugates. All synthesized systems were characterized by SEM which demonstrated the visual size, surface features, and surface textures of the polymer microparticles. Insulin-transferrin and calcitonin-transferrin conjugates were successfully synthesized using the protein crosslinker SPDP, binding the two proteins with a disulfide bond. The multi-step conjugation reactions used to create the conjugates were analyzed by the use of UV spectroscopy and HPLC to ensure the quality of the final products. In both conjugation reactions, the final product yield was found to be over 70%. The in vitro loading and release characteristics for insulin-transferrin and calcitonin-transferrin were separately investigated. By testing loading and release using a number of different polymer systems with different synthesis parameters, it was possible to optimize the hydrogel carriers for use with each of the conjugates independently. Upon optimization, the ideal system for use with insulin-transferrin and calcitonin-transferrin was found to be P(MAA-g-EG) microparticles of <75 microns formed using a PEGDMA1000 crosslinker and a 50:50 monomer-to-solvent ratio for both conjugates through separate optimization processes. This optimized polymer carrier was found to release upwards of 50% of loaded insulin-transferrin conjugate and near 90% of loaded calcitonin-transferrin conjugate. The insulin-transferrin conjugate was further evaluated through the use of cellular and animal models. Using cellular models, the insulin-transferrin conjugate was shown to increase transport relative to insulin by a factor of 7, achieving an apparent permeability of 37 x 10⁹ cm/s. Also, in the presence of polymer microparticles, the insulin-transferrin conjugate increased transport by a factor of 14 times relative to insulin, achieve an apparent permeability of 72.8 x 10⁹ cm/s. The presence of the microparticles near the cells was found to improve conjugate transport by nearly 100%. The preliminary animal studies verified the successful synthesis of the insulin-transferrin conjugate as well as demonstrated the bioactivity of the insulin portion of the molecule by achieving a drop in blood glucose level upon subcutaneous injection.Item Probing specificity of RNA : ribonucleoprotein interactions through in vitro selection(2004) Cox, James Colin, 1974-; Ellington, Andrew D.RNA binding proteins play a crucial role in normal cellular functions. However, little work has been successful in developing a code of recognition that may be exploited for the creation of novel RNA binding proteins. Research presented here has attempted to discern applicable specificity rules of the most commonly found RNA binding motif, the ribonucleoprotein (RNP) domain. I have employed in vitro selection to facilitate this, as it has been utilized in the past to discover the natural binding sequences of nucleic acid binding proteins. The process of in vitro selection results in the evolution of nucleic acid binding species, or aptamers. In order to perform these experiments within the span of my graduate career, I have developed an automated robotic workstation that is capable of performing in vitro selection. I have performed experiments that validate this automated system, and have further confirmed that it can successfully generate aptamers to protein targets possessing high specificity and affinity to their protein ligand. Moreover, this automated selection system has been able to recapitulate the natural sequence and structural specificity of the RNP-containing protein target used as a model system here, U1A. This evolutionarily conserved spliceosomal protein is chosen due to the extensive amount of biochemical and structural data available regarding binding to its cognate RNA. Twenty-one in vitro selection experiments have been executed in an automated manner against U1A point mutants. The results of these selections suggest that in vitro selection can be used as a tool by which determinants of specificity may be elucidated. Additionally, these selections have uncovered new information regarding U1A cognate specificity and affinity not currently known to the community.Item Protein engineering on soybean sterol methyl transferase leads to altered substrate binding and catalysis(Texas Tech University, 2004-12) Sinha, ArchanaSterol methyltransferases (SMTs) are ubiquitously represented in plants and they can serve as the rate-limiting enzymes in the 24-alkyl sterol (phytosterol) pathway. Together these enzymes are capable of converting sterol acceptors with a 24(25)-double bond (cycloartenol, CA; CI-activity) or 24(28)-double bond ((24)28-methyleneIophenol, ML; -C2-activity) in the sterol side chain into more that 60 distinct phytosterols in a single plant. Recently, we discovered using the soybean SMT that depending on the nature of the substrate olefin bound to SMT, either one or two catalytic reactions can proceed, concerted or step-wise, to generate the product diversity. To investigate the proposed role of aromatic amino acids that are part of a signature motif in the active site of SMT enzymes- F82YEYGWG88, Y83L and Y83F mutants were prepared and purified to homogeneity and the steady-state kinetic parameters were determined as described in this laboratory for the wild-type soybean SMT. The Y83L mutant performed much like the wild-type enzyme in terms of substrate acceptability, product distribution and physical property, but differences were detected in Y83F mutant. "When the mutant SMT activities were compared to the native SMT activities in relation to inhibition by 25- azacycloartenol (transition state analog) or 26,27-dehydrocycloartenol (mechanism based inhibitor), both sets of enzymes were found to be inhibited with equal efficacy, suggesting that the successive C-methylation of the Ä24 bond occurs at the same active center. Based on activity assays performed over the temperature range 15 to 40''C, the activation energy (Eact in KJ/mol) estimated from the Arrhenius plots were found to be: (i) wild-type, CA = 49, ML = 71; (ii) Y83F, CA = 65, ML = 52 and (iii) Y83L, CA = 98, ML =185. Analysis of the pH dependence of log kcat/Km for the wild-type and two mutants showed different profiles for Ä 24(25) and Ä24(28) -substrates. The results of the mutational and kinetic analyses are interpreted to imply that product diversity catalyzed by the soybean SMT is made possible by the relaxed control over substrate and intermediate conformations resulting from altered cation-ð interactions in the active sites of the mutant enzyme and relates to the different mechanisms catalyzed using different olefin substrates.Item Studies in pharmaceutical biotechnology : protein-protein interactions and beyond(2011-05) Umeda, Aiko; Zhang, Zhiwen Jonathan; Georgiou, George; Liu, Hung-wen; Johnson, Kenneth A.; Browning, Karen S.Pharmaceutical biotechnology has been emerging as a defined, increasingly important area of science dedicated to the discovery and delivery of drugs and therapies for the treatment of various human diseases. In contrast to the advancement in pharmaceutical biotechnology, current drug discovery efforts are facing unprecedented challenges. Difficulties in identifying novel drug targets and developing effective and safe drugs are closely related to the complexity of the network of interacting human proteins. Protein-protein interactions mediate virtually all cellular processes. Therefore both identification and understanding of protein-protein interactions are essential to the process of deciphering disease mechanisms and developing treatments. Unfortunately, our current knowledge and understanding of the human interactome is largely incomplete. Most of the unknown protein-protein interactions are expected to be weak and/or transient, hence are not easily identified. These unknown or uncharacterized interactions could affect the efficacy and toxicity of drug candidates, contributing to the high rate of failure. In an attempt to facilitate the ongoing efforts in drug discovery, we describe herein a series of novel methods and their applications addressing the broad topic of protein-protein interactions. We have developed a highly efficient site-specific protein cross-linking technology mediated by the genetically incorporated non-canonical amino acid L-DOPA to facilitate the identification and characterization of weak protein-protein interactions. We also established a protocol to incorporate L-DOPA into proteins in mammalian cells to enable in vivo site-specific protein cross-kinking. We then applied the DOPA-mediated cross-linking methodology to design a protein probe which can potentially serve as a diagnostic tool or a modulator of protein-protein interactions in vivo. To deliver such engineered proteins or other bioanalytical reagents into single live cells, we established a laser-assisted cellular nano-surgery protocol which would enable detailed observations of cell-to-cell variability and communication. Finally we investigated a possible experimental scheme to genetically evolve a fluorescent peptide, which has tremendous potential as a tool in cellular imaging and dynamic observation of protein-protein interactions in vivo. We aim to contribute to the discovery and development of new drugs and eventually to the overall health of our society by adding the technology above to the array of currently available bioanalytical tools.Item Transporter engineering as a tool for metabolic engineering(2013-08) Young, Eric Mosher; Alper, Hal S.; Appling, Dean; Contreras, Lydia; Georgiou, George; Maynard, JenniferThe purpose of metabolic engineering is to understand, design, and optimize metabolism. The objective is chemicals synthesis by microbes. To fulfill this purpose and achieve this objective, tools that control metabolism are essential. Molecular transport is a vital metabolic step yet tools to control it are underdeveloped. Therefore, this work aims to establish transporter engineering, a tool that can rewire transport. Efficient xylose utilization is a key component to economical consumption of lignocellulosic biomass, the most abundant source of sugars on the planet. Transport is a limiting step in the metabolism of xylose by the industrial yeast Saccharomyces cerevisiae. In yeast, transport proteins enabling xylose uptake also permit transit of a broad spectrum of other sugars. Furthermore, glucose is preferred as a substrate to the exclusion of xylose. Therefore, the goal of transporter engineering in this context is twofold: improve xylose uptake while reducing glucose uptake. Four strategies were used to accomplish this goal. First, we performed an iterative bioprospecting approach to explore the extant biodiversity of sugar transporters. However, the transporters tested lack efficient and exclusive xylose transport, motivating development of additional engineering strategies. Second, a directed evolution strategy increased xylose transport efficiency, demonstrating the power directed evolution has to improve transport phenotypes. Third, a targeted engineering strategy was used to analyze key residues responsible for the improved xylose transport phenotype, representing the first targeted engineering strategy to improve xylose growth and reduce glucose growth. Finally, rational engineering was explored. With all of the information collected using the previous strategies, design rules could be developed and implemented. A triple mutant of C. intermedia GXS1 was engineered that does not confer growth on glucose, but xylose growth is retained. By implementing this design rule in S. stipitis RGT2 and S. cerevisiae HXT7, additional xylose exclusive variants can be engineered. This demonstrates that a fundamental design component has been identified and can be used to rewire transport. Thus, this work builds the foundation for molecular transporter engineering.