Show simple item record

dc.contributor.advisorChaikof, Elliot L.
dc.contributor.advisorDesai, Michael M.
dc.contributor.advisorSpringer, Michael
dc.contributor.advisorHogle, James M.
dc.contributor.authorPacker, Michael Samuel
dc.date.accessioned2019-08-09T09:06:50Z
dash.embargo.terms2019-05-01
dc.date.created2017-05
dc.date.issued2017-04-17
dc.date.submitted2017
dc.identifier.citationPacker, Michael Samuel. 2017. Continuous Evolution of Proteases With Altered Specificity. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41141523*
dc.description.abstractThe following thesis work aims to establish a method for the generation of proteases with tailor-made substrate specificities. This programmability will enable the design of proteases that modulate the activity of a target protein of biotechnological or therapeutic relevance. In the first phase of this project, we developed and validated a system for the phage-assisted continuous evolution (PACE) of protease enzymes. In pilot feasibility studies, we used this system to evolve hepatitis C virus proteases that are resistant to small-molecule protease inhibitors. We then established that this PACE selection could also evolve specificity changes in two different model proteases: human rhinovirus (HRV) protease and tobacco etch virus (TEV) protease. After these proof-of-concept experiments, we began the phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target peptide sequence, HPLVGHM, that is present in IL-23, a cytokine implicated in inflammatory disease. A protease emerging from approximately 2,500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and inhibits IL-23 mediated signaling in cultured primary murine splenocytes. We characterized the substrate specificity of this evolved enzyme using a protease specificity profiling method, revealing a mixture of shifted and broadened specificity at the six positions in which the target amino acid sequence differed. On-going studies seek to expand the scope of protease PACE through: (1) the development of a negative selection scheme to ablate off-target proteolysis, (2) the adaptation of disulfide compatible E. coli strains for the PACE of human circulating proteases, and (3) the evolution Botulinum toxin proteases to enable the destruction of intracellular target proteins.
dc.description.sponsorshipBiophysics
dc.format.mimetypeapplication/pdf
dc.language.isoen
dash.licenseLAA
dc.subjectDirected Evolution, Proteases
dc.titleContinuous Evolution of Proteases With Altered Specificity
dc.typeThesis or Dissertation
dash.depositing.authorPacker, Michael Samuel
dash.embargo.until2019-05-01
dc.date.available2019-08-09T09:06:50Z
thesis.degree.date2017
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.levelDoctoral
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
thesis.degree.nameDoctor of Philosophy
dc.type.materialtext
thesis.degree.departmentBiophysics
thesis.degree.departmentBiophysics
dash.identifier.vireo
dash.author.emailmsp96@cornell.edu


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record