Browsing by Subject "Directed evolution"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Directed evolution of phosphotriesterase for detoxification of the nerve agent VX(Texas A&M University, 2006-10-30) Ghanem, Eman MohamedPhosphotriesterase (PTE) isolated from the soil bacterium Flavobacterium sp. is a member of the amidohydrolase superfamily. PTE catalyzes the hydrolysis of a broad spectrum of organophosphate triesters including the insecticide paraoxon, and the chemical warfare agents; GF, sarin, and soman. In addition, PTE has been shown to catalytically hydrolyze the lethal nerve agent, VX. However, the rate of VX hydrolysis is significantly slower. PTE was subjected to directed evolution studies to identify variants with enhanced activity towards VX hydrolysis. First generation libraries targeted amino acid residues in the substrate binding site. The H254A mutation displayed a 4-fold enhancement in kcat and a 2-fold enhancement in kcat/Km over wild type PTE. The double mutant H254Q/H257F was isolated from the second generation libraries and displayed a 10-fold enhancement in kcat and a 3-fold enhancement in kcat/Km. In addition, H254Q/H257F displayed a 9-fold enhancement in kcat/Km for the hydrolysis of the VX analog, demeton-S. An in vivo selection approach utilizing organophosphate triesters as the sole phosphorus source is discussed. The selection is based on co-expressing PTE with the phosphodiesterase (GpdQ) from E. aerogenes. Substrate specificity of GpdQ was investigated using a small library of structurally diverse organophosphate diesters and phosphonate monoesters. Results obtained from the in vivo growth assays showed that GpdQ enabled E. coli to utilize various organophosphate diesters and phosphonate monoesters as the sole phosphorus source. Cells co-expressing PTE and GpdQ were tested for their ability to utilize two different organophosphate triesters as the sole phosphorus source. The results from this experiment indicate that the growth rate is limited by the phosphotriesterase activity. Protein translocation to the periplasm was proven advantageous for in vivo selection since it overcomes the limitation of intercellular delivery of the substrate of interest. Translocation of PTE to the periplasmic space of E. coli was examined. Two signal peptides were tested; the native leader peptide from Flavobacterium sp. and the signal sequence of alkaline phosphatase. The results obtained from cellular fractionation indicated that neither signal peptides were able to translocate PTE to the periplasm and that the protein remained in the cytoplasm.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 Engineering the central dogma using emulsion based directed evolution(2016-05) Ellefson, Jared Wade; Ellington, Andrew D.; Barrick, Jeffrey; Marcotte, Edward; Georgoiu, George; Russell, RickThe central dogma of molecular biology forms the most basic (and fundamental) paradigm of how life operates. Despite its elegant simplicity, scientists are still uncovering enigmas of the central dogma - which has been shaped throughout billions of years of the Darwinian process. Even though the core concepts of the central dogma have largely been untouched by evolution (the universality of the genetic code, amino acid utilization, DNA/RNA base identity) scientific advances have shown that these fundamental properties can be altered dramatically. This implies the architectures of life are pliable and likely the result of extreme optimization and fine tuning of semi-random events that took place soon after the origin of life. Reengineering the parameters of life offers a unique way of testing evolutionary processes and perceived optimality of its components. Naturally, coaxing proteins and nucleic acids to function in an unnatural fashion is difficult. Development of techniques to enable these changes has relied heavily on the exploitation of water-in-oil emulsions (or, in vitro compartmentalization), which allows directed evolution at the single cell or even single molecule level. In particular, compartmentalized partnered replication (CPR) is a dual mode selection technique, coupling the in vivo functionality of a gene with the in vitro amplification via emulsion PCR. The CPR technique has enabled the development of synthetic promoter recognition by T7 RNA polymerase, unnatural amino acid incorporation by aminoacyl tRNA synthetase engineering, genetic code reassignment through tRNA evolution, and transcriptional regulation using repressors with novel allosteric effector molecules and operator binding sites. Using a similar technique, the template recognition of an Archaeal DNA polymerase was altered such that the polymerase utilizes both DNA and RNA templates with similar efficiencies. This resulted in a reverse transcriptase that can functionally proofread on RNA templates. These technologies will continue to play a pivotal role in the future development of particular aspects of the central dogma. As certain steps in this process are tweaked to have alternative functionalities and combined together, the gap between natural life and synthetically modified life widens and gives the Darwinian process of evolution new areas to explore.