Persistent Link:
http://hdl.handle.net/10150/194059
Title:
Non-Covalent Selection Methodologies Utilizing Phage Display
Author:
Meyer, Scott C.
Issue Date:
2007
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
In nature, non-covalent interactions are as important and dynamic as they are elusive. As such, the study of non-covalent interactions both in vivo and in vitro has proven to be challenging. Given the potential benefits of elucidating protein-protein, ligand-receptor, and other biologically relevant interactions, the development of methodologies for the study of non-covalent interactions is an attractive goal.Biologically encoded protein and peptide libraries that connect the genotypic information with the expressed phenotype have emerged in recent years as powerful methods for studying non-covalent interactions. One of the quintessential platforms for the creation of such libraries is phage display. In phage display, the connection between genetic information and the corresponding protein allows for the iterative isolation and amplification of library members that possess a desired function. Hence, an in vitro selection can be used to isolate epitopes that bind to desired targets or display specific attributes.We have sought to develop novel phage display methodologies that have the potential to expand the scope of this in vitro selection platform. Specifically, we developed a method for the non-covalent attachment of a small molecule ligand to a cyclic peptide library. This system localizes the phage display library to the ligand binding site, thus allowing for the translation of the selected cyclic peptides to a covalently tethered bivalent inhibitor.The first class of biological molecules that we chose to target with our methodology is the biologically and therapeutically important class of enzymes called protein kinases. In the first demonstration of this strategy, we were able to isolate cyclic peptide ligands for the model kinase PKA (cAMP-dependent protein kinase), which were subsequently translated to a bivalent inhibitor. This inhibitor showed both increased affinity and selectivity for PKA in relation to other protein kinases.In a separate project, we sought to develop a method for the isolation of small molecule-responsive mutants of a well-characterized protein scaffold from a phage display library. During these investigations, we discovered interesting homologous single-point mutations of the protein that resulted in large spherical oligomers that may mimic species relevant to the study of protein misfolding diseases such as Alzheimer's.
Type:
text; Electronic Dissertation
Keywords:
phage display; kinase; avidin; NeutrAvidin; PKA; cAMP-dependent protein kinase
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Ghosh, Indraneel

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleNon-Covalent Selection Methodologies Utilizing Phage Displayen_US
dc.creatorMeyer, Scott C.en_US
dc.contributor.authorMeyer, Scott C.en_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractIn nature, non-covalent interactions are as important and dynamic as they are elusive. As such, the study of non-covalent interactions both in vivo and in vitro has proven to be challenging. Given the potential benefits of elucidating protein-protein, ligand-receptor, and other biologically relevant interactions, the development of methodologies for the study of non-covalent interactions is an attractive goal.Biologically encoded protein and peptide libraries that connect the genotypic information with the expressed phenotype have emerged in recent years as powerful methods for studying non-covalent interactions. One of the quintessential platforms for the creation of such libraries is phage display. In phage display, the connection between genetic information and the corresponding protein allows for the iterative isolation and amplification of library members that possess a desired function. Hence, an in vitro selection can be used to isolate epitopes that bind to desired targets or display specific attributes.We have sought to develop novel phage display methodologies that have the potential to expand the scope of this in vitro selection platform. Specifically, we developed a method for the non-covalent attachment of a small molecule ligand to a cyclic peptide library. This system localizes the phage display library to the ligand binding site, thus allowing for the translation of the selected cyclic peptides to a covalently tethered bivalent inhibitor.The first class of biological molecules that we chose to target with our methodology is the biologically and therapeutically important class of enzymes called protein kinases. In the first demonstration of this strategy, we were able to isolate cyclic peptide ligands for the model kinase PKA (cAMP-dependent protein kinase), which were subsequently translated to a bivalent inhibitor. This inhibitor showed both increased affinity and selectivity for PKA in relation to other protein kinases.In a separate project, we sought to develop a method for the isolation of small molecule-responsive mutants of a well-characterized protein scaffold from a phage display library. During these investigations, we discovered interesting homologous single-point mutations of the protein that resulted in large spherical oligomers that may mimic species relevant to the study of protein misfolding diseases such as Alzheimer's.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectphage displayen_US
dc.subjectkinaseen_US
dc.subjectavidinen_US
dc.subjectNeutrAvidinen_US
dc.subjectPKAen_US
dc.subjectcAMP-dependent protein kinaseen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairGhosh, Indraneelen_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberCordes, Matthewen_US
dc.contributor.committeememberGlass, Richarden_US
dc.contributor.committeememberPolt, Robinen_US
dc.identifier.proquest2448en_US
dc.identifier.oclc659748368en_US
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