Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation
Enzymes are selective, operate efficiently in mild conditions and are biodegradable. Hence, enzymes have become an attractive option for a wide range of academic and industrial processes. However, enzymes are often labile at higher temperatures which can limit the operational range. Current enzyme s...
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ndltd-bl.uk-oai-ethos.bl.uk-7288012018-06-12T03:13:07ZEngineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formationSchoene, ChristopherHowarth, Mark2016Enzymes are selective, operate efficiently in mild conditions and are biodegradable. Hence, enzymes have become an attractive option for a wide range of academic and industrial processes. However, enzymes are often labile at higher temperatures which can limit the operational range. Current enzyme stabilization approaches can be time-intensive and often have only a minor effect. Enzyme cyclization has been suggested as an alternative and more general approach. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher and Isopeptag/Pilin-C are peptide/protein pairs capable of forming an irreversible isopeptide bond upon reconstitution. By genetically encoding SpyTag, SnoopTag and Pilin-C on the Nterminus and SpyCatcher, SnoopCatcher and Isopeptag on the C-terminus, I developed a method for the simple in vivo cyclization of enzymes. TEM-1 β-lactamase, which is prone to irreversible aggregation above 37 °C, remained soluble at temperatures up to 100 °C after isopeptide-mediated cyclization and upon cooling retained high catalytic activity. Differential scanning calorimetry showed that isopeptide-mediated cyclization had little effect on the Tm of the enzyme. Instead, isopeptide-mediated cyclization increased the reversibility of the denaturation. SpyTag/SpyCatcher was found to be the most effective pair for conferring thermal resilience and was also used to cyclize and increase the thermal resilience of dihydrofolate reductase and phytase. SpyTag/SpyCatcher-mediated cyclization had only minor effects on the thermal resilience of glucose oxidase and luciferase. Therefore, there is a limit to how general the effect of isopeptide-mediated cyclization is. To start addressing the limitations of the current approach, I developed a phage display-based strategy for the directed evolution of SpyTag/SpyCatcher reactivity. Isopeptide-mediated cyclization is a potentially powerful approach for the enhancement of protein resilience.University of Oxfordhttp://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.728801https://ora.ox.ac.uk/objects/uuid:88bc979a-b734-4f17-a7c7-47251fbefb42Electronic Thesis or Dissertation |
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Enzymes are selective, operate efficiently in mild conditions and are biodegradable. Hence, enzymes have become an attractive option for a wide range of academic and industrial processes. However, enzymes are often labile at higher temperatures which can limit the operational range. Current enzyme stabilization approaches can be time-intensive and often have only a minor effect. Enzyme cyclization has been suggested as an alternative and more general approach. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher and Isopeptag/Pilin-C are peptide/protein pairs capable of forming an irreversible isopeptide bond upon reconstitution. By genetically encoding SpyTag, SnoopTag and Pilin-C on the Nterminus and SpyCatcher, SnoopCatcher and Isopeptag on the C-terminus, I developed a method for the simple in vivo cyclization of enzymes. TEM-1 β-lactamase, which is prone to irreversible aggregation above 37 °C, remained soluble at temperatures up to 100 °C after isopeptide-mediated cyclization and upon cooling retained high catalytic activity. Differential scanning calorimetry showed that isopeptide-mediated cyclization had little effect on the Tm of the enzyme. Instead, isopeptide-mediated cyclization increased the reversibility of the denaturation. SpyTag/SpyCatcher was found to be the most effective pair for conferring thermal resilience and was also used to cyclize and increase the thermal resilience of dihydrofolate reductase and phytase. SpyTag/SpyCatcher-mediated cyclization had only minor effects on the thermal resilience of glucose oxidase and luciferase. Therefore, there is a limit to how general the effect of isopeptide-mediated cyclization is. To start addressing the limitations of the current approach, I developed a phage display-based strategy for the directed evolution of SpyTag/SpyCatcher reactivity. Isopeptide-mediated cyclization is a potentially powerful approach for the enhancement of protein resilience. |
author2 |
Howarth, Mark |
author_facet |
Howarth, Mark Schoene, Christopher |
author |
Schoene, Christopher |
spellingShingle |
Schoene, Christopher Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
author_sort |
Schoene, Christopher |
title |
Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
title_short |
Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
title_full |
Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
title_fullStr |
Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
title_full_unstemmed |
Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
title_sort |
engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation |
publisher |
University of Oxford |
publishDate |
2016 |
url |
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.728801 |
work_keys_str_mv |
AT schoenechristopher engineeringrobustenzymesviaheadtotailcyclizationusingspontaneousisopeptidebondformation |
_version_ |
1718693628572860416 |