Browsing by Subject "library design"

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    Constrained Diversification Enhances Protein Ligand Discovery and Evolution
    (2017-04) Woldring, Daniel
    Engineered proteins have strongly benefited the effectiveness and variety of precision drugs, molecular diagnostic agents, and fundamental research reagents. A growing demand for new therapeutics motivates the innovative use of natural proteins – improving upon their native properties – as well as discovering proteins with entirely new functionality. Importantly, these are fundamentally separate goals. While evolving improved function can result from making a few carefully chosen mutations, discovering novel function often requires giant leaps to be taken in protein sequence space. Discovering novel function is a notoriously challenging task. The immensity of sequence space (e.g. proteins of length n have 20^n unique options) makes it essentially impossible to experimentally or computationally test all possible protein sequences. Within this space, the landscape is incredibly barren and rugged (i.e. most sequences lack function entirely and making small changes to a protein often damage the activity). Rather than randomly mutating a protein, combinatorial protein libraries provide a systematic and efficient approach for searching sequence space. This method offers precise control over which protein sites are mutated and which amino acids are allowed at the diversified sites. To improve the likelihood of sampling useful sequences, numerous techniques can elucidate the structure-function relationships in proteins. Generally, these techniques have not been applied to combinatorial library design; however, we propose that some, or all, could be greatly beneficial in this area. In this thesis work, protein libraries are designed for the purpose of discovering high affinity, specific binders to a collection of interesting targets. High-throughput sequencing of evolved binders, natural protein-protein interface composition, structural assessment, and computational analysis of stability upon mutation collectively informed sitewise library designs – residues predicted to support function were allowed but destabilizing residues or those not likely to benefit function were avoided. We use multiple small protein scaffolds (affibody and fibronectin) as model systems to test the hypothesis that constrained sitewise diversity will improve the efficiency of novel protein discovery. This hypothesis was experimentally supported by a direct comparison of high-affinity ligand discovery between the sitewise constrained library and a uniformly diversified library (i.e. allowing all 20 residues at each diversified site). The constrained library showed a 13-fold improved likelihood of binder discovery. Moreover, the constrained library variants demonstrated superior thermal stability (Tm 15 °C higher than unbiased variants). This work provides further evidence that sitewise diversification of protein scaffolds can improve the overall quality of combinatorial libraries by offering broad coverage of sequence space without sacrificing stability.
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    Protease Engineering To Enable Noninvasive Disease Detection
    (2020-03) Mikolajczyk, Brian
    Proteases are proteolytic enzymes with a wide range of industrial, biotechnological, and medical applications. Due to their importance, proteases have been the subject of many attempts to engineer improved performance, but campaigns to improve activity via directed evolution have been hindered by inefficient analytical techniques and insufficient understanding of sequence-function relationships. Tobacco etch virus protease (TEVp) has mostly been engineered for attributes other than catalytic activity, and most of the past efforts have employed random mutagenesis methods such as error-prone PCR as opposed to targeted mutagenesis. We developed a novel and seemingly generalizable yeast surface display approach that co-displays protease mutants adjacent to substrate on the same Aga2 anchor protein. Enhanced activity mutants are identified by protease cleavage of tethered substrate removing an epitope tag, which empowers flow cytometric isolation of cells with a decrease in anti-epitope antibody signal. This platform was shown to quantitatively differentiate catalytic activity at the single-cell level for TEVp and sortase A. We leveraged this display platform to perform high throughput screens on seven structure-based active site combinatorial libraries created via saturation mutagenesis, and then screened a second-generation library combining the resultant beneficial mutations. Deep sequencing of functional mutants elucidated sequence-function relationships across 34 sites and identified improved multi-mutants. Clonal analysis of a host of recombinant TEVp multi-mutants with purified substrate demonstrated up to 2.9-fold improvement in catalytic efficiency, generally via decreased KM. The novel yeast surface protease/substrate co-display system and the insights gleaned on rational active site library design and the TEVp sequence-function map will aid future protease engineering efforts, and the collection of improved multi-mutants will benefit the biotechnological community in utilizing TEVp in its multitude of applications. One class of application for engineered proteases is physiological release of diagnostic or therapeutic moieties. We introduced a novel extension of synthetic reporters to noninvasively detect abnormal receptor expression. Synthetic reporters have been demonstrated to noninvasively detect a host of diseases via nanoparticles conjugated to reporters via substrate linkers; biomarkers are generated dependent upon a disease-specific enzyme and filtered into the urine. This approach is limited by its reliance on upregulation of disease-specific proteases, but many diseases are characterized by abnormal expression of cell-surface receptors. The new approach harnesses ligand-enzyme fusion proteins to impart exogenous enzymatic activity to tissue with aberrant receptor expression. A mathematical model for epidermal growth factor receptor (EGFR) tumor xenografts in mice demonstrated feasibility of this approach with TEVp-based fusions, suggesting detection down to tumor diameters of 0.28 mm at standard substrate concentrations. Multiple fusions were produced using different enzymes, ligands, and orientations, and binding and catalytic activity was generally well preserved, indicating a modular fusion framework. Demonstrating feasibility with anti-EGFR TEVp-based fusions in an in vitro cellular assay was not consistently successful. However, the following limitations were identified for improvement: high substrate lability, and insufficient fusion-specific product generation due to inadequate catalytic activity – which would motivate protease engineering – or suboptimal fusion linker design that resulted in ineffective projection of receptor-bound fusion’s enzyme component to engage soluble substrate. Together, this work introduced a novel extension of the synthetic reporter concept to quantify receptor expression, and we have demonstrated theoretical in vivo feasibility as well as empirical functionality of the required ligand-enzyme fusions. We have also introduced a novel display platform that can be harnessed for screening combinatorial protease libraries to find mutants with improved catalytic efficiency, which will aid the synthetic reporter approach.