Engineering 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. / text

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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 stabilit / text

The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.

Biofilms are formed in aquatic environments by the attachment of bacteria to submerged surfaces, to the air/liquid interface, and to each other. Although biofilms are associated with disease and biofouling, the robust nature of biofilms; for example, their ability to tolerate chemical and physical stresses, makes them attractive for beneficial biotechnology applications such as bioremediation and biofuels. Based on an understanding of diverse signals and regulatory networks during biofilm development, biofilms can be engineered for these applications by manipulating extracellular/intercellular signals and regulators. Here, we rewired the global regulator H-NS of Escherichia coli to control biofilm formation using random protein engineering. H-NS variant K57N was obtained that reduces biofilm formation 10-fold compared with wild-type H-NS (wild-type H-NS increases biofilm formation whereas H-NS K57N reduces it) via its interaction with the nucleoid-associated proteins Cnu and StpA. H-NS K57N leads to enhanced excision of the defective prophage Rac and results in cell lysis through the activation of a host killing toxin HokD. We also engineered another global regulator, Hha, which interacts with H-NS, to disperse biofilms. Hha variant Hha13D6 was obtained that causes nearly complete biofilm dispersal by increasing cell death by the activation of proteases. Bacterial quorum sensing (QS) systems are important components of a wide variety of engineered biological devices, since autoinducers are useful as input signals because they are small, diffuse freely in aqueous media, and are easily taken up by cells. To demonstrate that biofilms may be controlled for biotechnological applications such as biorefineries, we constructed a synthetic biofilm engineering circuit to manipulate biofilm formation. By using a population-driven QS switch based on the LasI/LasR system and biofilm dispersal proteins Hha13D6 and BdcAE50Q (disperses biofilms by titrating cyclic diguanylate), we displaced an existing biofilm and then removed the second biofilm. Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Here, we engineered toxin MqsR with greater toxicity and demonstrated that the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems.

The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.

Errata and corrections pasted on front end paper. / Copies of three of author's previously published articles contained in back pocket. / Bibliography: leaves 178-219. / ix, 219, [130] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis aims to generate a library of chromosomally derived transcriptional promoter ::lacZ fusion mutants in an environmental isolate of L. monocytogenes (DRDC8). A lacZ transcriptional fusion mutant that displayed increased B-galactosidase activity in response to the calcium chelater EGTA is investigated in detail. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997

Diss. (sammanfattning) Umeå : Umeå universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 148-157).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [161]-169).