Browsing by Subject "Enzyme engineering"
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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 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 stabilit